University of Bath

PHD

Phospholipid composition of Saccharomyces cerevisiae and Zygosaccharomycesbailii and their response to sulphur dioxide

Pilkington, Bridget Jane

Award date:1989

Awarding institution:University of Bath

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PHOSPHOLIPID COMPOSITION OF SACCHAROMYCES CEREVISIAE

AND ZYGOSACCHAROMYCES BAILII AND THEIR RESPONSE

TO SULPHUR DIOXIDE

Submitted by Bridget Jane Pilkington

For the Degree of Ph.D. of

The University of Bath

1989

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CONTENTS

Page No.

SUMMARY v

ACKNOWLEDGEMENTS vii

INTRODUCTION 1

SULPHUR DIOXIDE 1

Properties of sulphur dioxide in solution 2

Reactivity of sulphur dioxide 4

Sulphite-binding compounds 7

Antimicrobial activity of sulphur dioxide 9

Application and treatment concentrations of

sulphiting agents 10Hazards of using sulphiting agents 11

YEASTS AND FOOD SPOILAGE 15

Spoilage yeasts 15

Mechanisms of action of sulphur dioxide

on yeasts 17

Sulphur dioxide transport 17

Intracellular effects of sulphur dioxide 19

Sulphur dioxide targets 21

Stimulation of the production of

sulphite-binding compounds 25

Resistance to sulphur dioxide 27

YEAST PLASMA MEMBRANE: COMPOSITION AND FUNCTION 29

Structure of the plasma membrane 38

Plasma membrane composition and diffusion 42

ii.

Page No.

METHODS 50

ORGANISMS 50

EXPERIMENTAL CULTURES 50

ASSESSMENT OF SULPHUR DIOXIDE TOLERANCE 52

MEASUREMENT OF SULPHITE ACCUMULATION 53

MEASUREMENT OF PLASMA-MEMBRANE AREA IN ORGANISMS 54

MEASUREMENT OF INTRACELLULAR WATER VOLUME 55

MEASUREMENT OF INTRACELLULAR pH VALUES 56

(a) Use of propionic acid 56

(b) Use of fluorescein diacetate as a

fluorescent probe 58

VIABILITY MEASUREMENTS 59

ANALYTICAL METHODS 59

(a) Free sulphite 59

(b) Pyruvate 60

(c) Acetaldehyde 61

(d) Glycerol 61

(e) Ethanol 62

LIPID ANALYSIS 62

(a) Lipid extraction 62

(b) Fatty-acyl composition of total cellular

phospholipids 64

(c) Fatty-acyl composition of individual

phospholipid classes 64

(d) Analysis of total cellular phospholipids 65

iii.

Page No.

MATERIALS 66

RESULTS 67

GROWTH OF ORGANISMS UNDER AEROBIC CONDITIONS 67

EFFECTS OF SULPHITE ON AEROBIC GROWTH 67

ACCUMULATION OF SULPHITE UNDER AEROBIC CONDITIONS 71

EFFECT OF SULPHITE ON YEAST VIABILITY 71

EFFECTS OF SULPHITE UPON INTRACELLULAR pH VALUES 71

PRODUCTION OF BINDING COMPOUNDS BY ORGANISMS

GROWN AEROBICALLY IN THE PRESENCE OF SULPHITE 77

FATTY-ACYL COMPOSITION OF PHOSPHOLIPIDS FROM

AEROBICALLY-GROWN YEASTS 86

GROWTH OF SACCHAROMYCES CEREVISIAE NCYC 431 UNDER

ANAEROBIC CONDITIONS 95

FATTY-ACYL COMPOSITION OF PHOSPHOLIPIDS FROM

ANAEROBICALLY-GROWN YEASTS 97

EFFECT OF FATTY-ACYL UNSATURATION AND CHAIN LENGTH

ON PERMEATION OF SULPHITE INTO YEASTS 99

DISCUSSION 113

SCREENING FOR SULPHITE TOLERANCE IN YEASTS 113

INITIAL EFFECTS OF SULPHITE ACCUMULATION IN YEASTS 114

Sulphur dioxide transport 114

Intracellular water volumes and intracellular

pH values 116

iv.

Page No.

LONG TERM EFFECTS OF SULPHITE 122

Stimulation of acetaldehyde production 122

PLASMA-MEMBRANE COMPOSITION AND THE DIFFUSION OF

SULPHUR DIOXIDE INTO YEASTS 125

Plasma-membrane composition of aerobically

grown yeasts 125

Plasma-membrane composition of anaerobically

grown yeasts 127

Diffusion of sulphur dioxide and plasma-membrane

composition 130

REFERENCES 140

APPENDIX 173

SUMMARY

Sulphite inhibited growth of all four yeasts studied,

Zygosaccharomyces bailii NCYC 563 being the most sensitive and

Saccharomyces cerevisiae NCYC 431 the least. Vertical Woolf-Eadie35plots were obtained for initial velocities of S accumulation by

all four yeasts suspended in high concentrations of sulphite.35Equilibrium levels of S accumulation were reached somewhat faster

with strains of Sacch. cerevisiae than those with Zygosacch.35bailii. With all four yeasts, the greater the extent of S

accumulation, the larger was the decline in internal pH value.

Growth of Sacch. cerevisiae TC8 and Zygosacch. bailii NCYC 563, butto a lesser extent of Sacch. cerevisiae NCYC 431 and Zygosacch.

bailii NCYC 1427, was inhibited when mid-exponential phase cultures

were supplemented with 1.0 or 2.0 mM-sulphite, the decrease in

growth being accompanied by a decline in ethanol and pyruvate

production. Unless growth was completely inhibited, the

sulphite-induced decline in growth was accompanied by production of

acetaldehyde and additional glycerol.

Analyses were made of the total cellular phospholipids from all

four yeasts grown aerobically. Fatty-acyl residues of C1c C,_16:1 18:1and C^0.q predominated in phospholipids from Sacch. cerevisiae,while phospholipids from Zygosacch. bailii contained mainly C„^— 18! 2C1Q . and C,_ _ residues. Strains of Sacch. cerevisiae were found lo: l lo:u ----- -----------to contain higher contents of phospholipid (mg dry wt organisms

compared with strains of Zygosacch. bailii but proportions of

phospholipid classes were similar among each strain.

Phosphatidylcholine was the most common class of phospholipid

followed by phosphatidylethanolamine and phosphatidylinositol with

less than 10% as phosphatidylserine.Saccharomyces cerevisiae NCYC 431 grown anaerobically in media

supplemented with ergosterol and C c18.1» Ci8-2* C18*3or fatty acids contained phospholipids enriched with residues

of the exogenously provided acids, to a greater extent with shorter

chain than longer chain acids. In these organisms direct

correlation between mean fatty-acyl chain lengths and degree of

unsaturation (expressed as Amol value) of cellular phospholipids

indicated strict control of plasma-membrane synthesis and

maintenance of the fluidity and rigidity necessary for normal

plasma-membrane function. However, the proportions of each class of

phospholipid were not affected significantly by the change in

growth conditions. Plots of the permeability coefficient of SO^

accumulation, derived from Woolf-Eadie plots, against the degree of

unsaturation in phospholipids showed that the coefficient was

greater the lower the degree of unsaturation in the phospholipids.

There was no correlation between the mean fatty-acyl chain lengths

and permeability coefficients of SO^ accumulation in organisms but

there was very good correlation between the coefficient and the

ratio of mean fatty-acyl chain length and degree of unsaturation of

cellular phospholipids. It is concluded that permeability of the

yeast plasma membrane to SO^ is proportional to the thickness and

degree of fluidity of the plasma membrane.

ACKNOWLEDGEMENTS

I would like to express my thanks to my supervisor Professor

Anthony H. Rose for his help and guidance throughout the duration

of this project. My thanks are also due to the Agriculture and Food

Research Council for the provision of a research assistantship and

to the University of Bath for the opportunity to submit this work

for the degree of Ph.D.

INTRODUCTION

SULPHUR DIOXIDE

Sulphiting agents in various forms have enjoyed a long history

as food preservatives dating back to Roman times where wine vessels

were apparently sanitised with sulphur dioxide (Roberts and

McWeeny, 1972). One of the earliest reports of its use as a food

preservative dates to at least 1664 where cider was added to flasks

while they still contained sulphur dioxide (Evelyn, 1664). Although

no human ailment or untoward effect resulting from such use has

been recognised, concern over possible hazard goes back a

considerable length of time to an article published by Kionka in

1896 on the possible toxicity of sulphites in foods.

Nowadays sulphiting agents are widely used in foods and

beverages and applied in many chemical forms. The principal

compound used to generate sulphur dioxide and the related anions in

the preservation of foods and beverages is sodium metabisulphite

(Na-S_0_), designated additive E223 in Directives of the Europeand bEconomic Community (Hanssen and Marsden, 1984). Other compounds

frequently employed as sulphiting agents include gaseous sulphur

dioxide (SO ), potassium bisulphite (KHSO^), potassium

metabisulphite (K_S_0_), sodium bisulphite (NaHSCL) and sodium d. d. b 3

sulphite (Na^SOg). Their common characteristic is their ability to

release free molecular sulphur dioxide and it is this fraction that

is believed to be the active food preservative. The antimicrobial

activity of each compound varies according to its ability to

liberate sulphur dioxide and is expressed in terms of "sulphur

dioxide equivalents", i.e. stoicheiometric amounts of sulphur

dioxide available from each sulphiting agent.

Sulphiting agents are very successful preservatives not only

because of their antimicrobial properties. They are commonly used

to stop enzymic and non-enzymic browning, to act as anti-oxidants

and reducing agents, bleaching agents and general aids to food

processing. They also fulfil the basic criteria of being water

soluble, tasteless, odourless and generally recognised as non-toxic

in low concentrations. However, in the interests of the consumer

and manufacturers, more efficient and safer alternatives are being

sought, but to date none has been found. Possible alternatives

usually provide a narrower range of benefits, are often less

effective and nearly always more expensive.

Properties of Sulphur Dioxide in SolutionThe terminology in this field of research is sometimes confused

and needs to be clarified. The terms sulphite, bisulphite and

sulphur dioxide are often used interchangeably if not incorrectly.

This area is made more complicated because sulphite can become

bound to organic molecules so that it is necessary to specify

exactly what fraction is being considered. In solution,

metabisulphite generates sulphur dioxide, bisulphite and sulphite

anions. The proportion of these species present depends on the pH

value of the solution. The equilibria are:

so2 + h2o (H2S03) ^ HS03 + H+ ^ S032 + 2H+

sulphurdioxide

sulphurous bisulphite sulphite acid

The existence of sulphurous acid is largely unaccepted since

ultraviolet and infrared Raman spectroscopy have failed to reveal

its presence. Falk and Guiguere (1958) suggested that, in the

absence of stable sulphurous acid molecules in solution, SO^ is

dissolved in the molecular state and exists as SO^.H^O.

Dissociation constants for each of the two remaining equilibria

have been determined at low sulphite concentrations of the order of

those used as food preservatives. The reaction leading to the

ionisation of SO^ has a pKa value of 1.77 at 25°C, while the value

for the reaction leading to production of the sulphite ion under

the same conditions is 7.20 (King et al., 1981). Using these pKa

values, calculations have been made of the proportions of each

species present in solution as a function of pH value (Table 1).

Table 1. Percentage distribution of molecular species of sulphur

dioxide as a function of pH values. From King et al.

(1981).

Percentage of ■2-2“ HS03~ ““3pH value S0..H.0 HS0o SO,2

2.0 37.03 62.97 0

3.0 5.56 94.43 0.006

4.0 0.59 99.35 0.063

5.0 0.058 99.31 0.63

6.0 0.006 94.15 5.84

7.0 0.0002 61.30 38.70

Although widely different values of pKa for SO^ were found in the

literature, the more recent publication by Wedzicha (1984) supports

the values of King et al. (1981) with values of pKa 1.86 (Huss and

Eckert, 1977) and pKa 7.18 (Betts and Voss, 1970), respectively.

The antimicrobial activity of sulphiting agents increases inversely

with pH value where proportionally more molecular SO^ exists

(Macris and Markakis, 1974). Sulphite, like other weak-acid

preservatives e.g. benzoic and sorbic acids, exhibits the highest

antimicrobial action with the undissociated form of the acid

(Eklund, 1983). Ionised species show no significant antimicrobial

activity (Ingram, 1959; Carr et al., 1976). From a practical

viewpoint, the pKa value of sulphite defines the pH range over

which it may be expected to be effective as an antimicrobial agent

and this is why sulphur dioxide is the preservative of choice for

foods and beverages of a low pH value (Sinskey, 1980).

Reactivity of Sulphur Dioxide

Analysis and control of sulphite residues in foods is made

complicated by the rapid reactions between sulphiting agents and a

variety of food components. All three species that are found in

solutions of sulphite, especially the bisulphite ion, are

chemically very reactive. Sulphites react readily with reducing

sugars, compounds containing carbonyl groups and proteins to form

sulphite addition compounds. Aqueous sulphur dioxide solutions

react readily with aldehydes and more slowly with ketones to

produce a-hydroxysulphonates (Joslyn and Braverman, 1954):

5.

CH3\

CH. OH

C=0 + H

H/ H S03acetaldehyde sulphite a-hydroxysulphonate

Combination of sulphite with cyclic sugars is slower than with

open-chain aldehydes. Ingram and Vas (1950) showed that galactose,

mannose and arabinose quickly form addition compounds with

sulphite; maltose, lactose and glucose are less active while

sucrose and fructose are largely inactive. They prepared a 0.5%

(w/v) solution of sodium sulphite containing 1.0% (w/v) citric acid monohydrate. Sugars (5% w/v) were added and allowed to stand at

room temperature for 24 hours. After that time, the percentage of

combined sulphite in each of the solutions were 88, 68, 63 and 20 for arabinose, mannose, galactose and glucose, respectively. The

significant sulphite-binding capacity of glucose has encouraged

experimenters to favour using fructose which has a minimal

sulphite-binding capacity in physiological investigations (Warth,

1986).

Burroughs and Sparks (1973a) identified 11 different sulphite-

binding compounds in cider, but the major portion (59-77%) of the

bound SO^ was attributed to complexes with just three of these,

namely acetaldehyde, pyruvate and 2-oxoglutaric .acid. The rate of

formation of sulphite-binding adducts is dependent on the

concentration of binding compound, pH value and temperature (Rehm,

1964; Burroughs and Sparks, 1973c).

In the presence of molecular oxygen sulphite will rapidly

oxidise, the stoicheiometric equation for which is:

so32- + yzo2 - so42~

Bisulphite, however, is much less easily oxidised by oxygen. Data

for this reaction are thoroughly reviewed by Wedzicha (1984).

Another reaction of significance is that between bisulphite and

disulphide bonds (Means and Feeney, 1971; Ough, 1983):

R-S-S-R + SO 2“ =? R-S-S-0 ~ + RS~O J

The products of the reaction are thiosulphonates sometimes known as

Bunte salts. Disulphide bonds lying between juxtaposed cysteine

residues help to stabilize the tertiary structure of proteins

essential for normal enzymic activity. This may be a clue in

helping to understand sulphite's antimicrobial properties leading

to conformational changes in proteins and causing loss of enzyme

function.

A review by Ough (1983) reports on how thiamin pyrophosphate, a

required enzymic cofactor in many reactions, can be destroyed by

sulphite, and excess SO^ can, by sulphitolysis of thiamin, destroy

the nutritive value of thiamin potentially resulting in vitamin B

deficiency (Williams et al., 1935; Gunnison, 1981).

Interactions of sulphiting agents with nucleic acids causing

mutagenesis have been reported (Hayatsu and Miura, 1970; Mukai

et al., 1970; Shapiro et al., 1973). These and other interactions

with SO^ are well documented in reviews by Hammond and Carr (1976)

and Wedzicha (1984).

Sulphite-Binding CompoundsWhen sulphite is added as a preservative to fruit juices, wines

and ciders etc., part of it combines more or less rapidly with

various carbonyl compounds some of which will be present in the

extracellular media, food or beverage, and some produced by

contaminating organisms or fermentation yeasts. As it is largely

accepted that the bound species have little or no antimicrobial

activity, the bound preservative is effectively lost and in

combination with auto-oxidation of sulphite, serves to lower

dramatically the efficiency of sulphiting agents. Identification of

such binding compounds is therefore of great practical and

commercial interest when considering optimising the effect of SO^.

Acetaldehyde has long been recognised as the major sulphite-

binding compound in most wines with glucose generally having little

effect, whereas some wines derived from grapes affected by mould

growth have exceptionally high sulphite-binding power due to

unidentified substances. Kielhofer and Wurdig (1960) designated the

fraction of sulphite bound to compounds other than acetaldehyde or

glucose as "Rest" or residual SO^.

Burroughs and Sparks (1964a) identified and isolated three

sugars, namely glucose, xylose and xylosone, responsible for

binding most of the sulphite in uncontaminated fruit juice. In

cider, the same compounds are accompanied by arabinose and

galacturonic acid, derived from the degradation of pectin, and the

8.

products of fermentation, namely acetaldehyde, pyruvate and

2-oxoglutarate. In the presence of spoilage organisms, the list of

potential sulphite-binding compounds grows longer with more

carbonyl compounds being produced. The very high sulphite-binding

power of juices and ciders from damaged fruit has been traced to

the combined activities of moulds and acetic-acid bacteria, chiefly

Acetomonas species, resulting in high concentrations of sulphite-

binding compounds including 5-fructulose, 2-oxogluconic and

2,5-di-oxogluconic acids (Burroughs and Sparks, 1962-1963). All of

these observations emphasise the need to minimise the inclusion of

potential binding compounds in products in order to maximise the

efficiency of sulphiting agents. Burroughs and Sparks (1973a,

1973b) went on to identify and determine dissociation constants for

a number of common carbonyl-bisulphite compounds in wines and

ciders (Table 2).

Table 2. Apparent equilibrium constants of a-hydroxysulphonates.

Adapted from Burroughs and Sparks (1973a)

Carbonyl compound Concentration (mM) of Equilibrium constantCarbonyl Total SO at

- compound pH 3.0 pH 4.0

Acetaldehyde 6.0 4.0 1.5.10-6 1.4.10"62,5-Di-oxogluconic acid 2.0 0.6- 7.2 -44.5.10 -44.3.10

Galacturonic acid 10.0 8.0-20.0 1.6.10“2 2.1.10“22-0xoglutaric acid 2.0 2.0-10.0 4.9.10~4 7.0.10’45-Fructulose 2.0 1.2- 7.5 -43.4.10 -43.3.10

Pyruvic acid 2.0 0.8- 5.0 -41.4.10 -42.2.10L-Xylosone 2.0 2.0-10.0 1.4.10”3 1.4.10"3

Combination of sulphite with carbonyl compounds is reversible

to a greater or lesser extent depending upon their respective

equilibrium constants; products are therefore essentially buffered

with respect to sulphite. Acetaldehyde has a very low dissociation

constant and has a strong affinity for sulphite so that, even in

the presence of low concentrations of sulphite, nearly all of the

acetaldehyde becomes bound whereas other compounds bind

progressively as sulphite concentrations increase.

Antimicrobial Activity of Sulphur DioxideCommercially sulphiting agents are used in more acidic foods

and beverages to prevent the growth of (a) acetic acid-producing

and malo-lactic bacteria, (b) fermentation and food-spoilage

yeasts, (c) fruit moulds (Joslyn and Braverman, 1954). Sulphites

are more effective in inhibiting bacterial and mould contamination

than that caused by yeasts, species of which show a considerable

range of tolerance to SO^. The selective nature of SO^ enhances its

value in control of undesirable fermentation and contamination in

wine making.

Free molecular SO^ is the active form of the sulphiting agents

in terms of antimicrobial action. Bound forms generally have

minimal antimicrobial activity (Rehm, 1964). Molecular SO^ is more

than 1000 times as active as the bisulphite or sulphite ion against Escherichia coli, 500 times more effective against yeasts and 100

times more effective against Aspergillus niger (Rehm and Wittman,

1962). Reports of the antimicrobial properties of bound SO^,

reviewed by Beech and Thomas (1985), suggest that antimicrobial

activity attributed to bound SO^ probably arises as the bound

complex, e.g. pyruvate-sulphite, is metabolised releasing free SO ,

or simply by virtue of the dynamic equilibrium in existence between

the bound and free species giving rise to SO^. Stratford and Rose

(1985) showed the former to be true. In Saccharomyces cerevisiae

TC8 radiolabelled sulphite derived from a pyruvate-sulphite complex was taken up into organisms more quickly than pyruvate, strongly

suggesting that dissociation of the complex takes place before its

components are transported by organisms.

Application and Treatment Concentrations of Sulphiting AgentsConcentrations of sulphur dioxide used commercially vary

greatly according to the products, ranging between zero and 3000

ppm (SO^ equivalents) on a dry-weight basis. Dehydrated fruits,

such as apples, apricots and peaches, are treated to contain the

greatest amount in this range. Dehydrated vegetables and prepared

soup mixtures range between a few hundred and 2000 ppm. A

World-wide average for wines would be about 100-400 ppm with about

2-8 ppm in beers. It should be noted that concentrations of

sulphites used in some products are self-limiting because of

organoleptic considerations. Different treatment concentrations are

required with various sulphiting agents to yield equivalent doses

of active agent (Modderman, 1986). For comparative purposes it is

helpful to calculate treatment concentrations on the basis of

percentage theoretical yield of SO , e.g. for the sulphiting agents

sulphur dioxide, sodium bisulphite, sodium metabisulphite,

potassium metabisulphite and potassium bisulphite percentage

theoretical yields of SO^ are 100, 61.56, 67.39, 57.60 and 53.32%,

respectively (Green, 1976). It should be noted that these

concentrations are rarely achieved and can only be used as a guide.

Yields will be dependent upon the solubilities of each species and

physical constraints put upon the equilibria by conditions such as

temperature, pH value, pressure and, of course, the presence of

sulphite-binding compounds.

Sulphite is naturally produced from sulphate during the

fermentation process as an intermediate in the biosynthesis of the

sulphur-containing amino acids cysteine and methionine in yeasts

(Institute of Food Technologist's Expert Panel on Food Safety and

Nutrition and the Committee on Public Information, 1975; Brewer and

Fenton, 1980; Ough, 1983). Wurdig and Schlotter (1968) reported

yeast strains capable of producing up to 130 ppm of SO^ in

fermentation broths.

One associated problem with sulphiting is that concentrations

exceeding 50 ppm or 0.8 mM free SO^ can impart undesirable flavours

and odours to the product (Taylor et al., 1986). Since a large

proportion of this can be generated by fermentation yeasts before

sulphite addition, it is necessary to control sulphite levels

(Garza-Ulloa, 1980; Warner et al. , 1987). Both free and bound

concentrations of SO^ are measured throughout production and

processing of foods, but the concentrations at the point of

consumption can only be estimated since little is known of the

effects of storage upon sulphites. Generally SO^ concentrations

fall during storage, and rapidly by auto-oxidation if exposed to

air. Associated problems of measuring sulphite concentrations while

minimising loss of sulphur dioxide were recorded by Mason and Walsh

(1928). Postgate (1963) later observed that a 0.1 M-sulphite

solution in physiological saline shaken in air at 37°C fell to

0.07 M after one hour and to 0.022 M after 2.5 hours. Actual

concentrations of free and total SO^ remaining in a particular food

product are dictated by the extent of absorption of the sulphites

during treatment, the nature of the processing treatment following

sulphite addition, and the conditions of storage (Schroeter, 1966).

The efficiency of sulphiting agents can be increased fairly

simply and economically. For example, in the cider industry, it is

essential to select a fermenting yeast that does not produce

excessive amounts of sulphite-binding compound (Burroughs and

Whiting, 1961) and is a poor sulphite producer (Eschenbruch and

Bonish, 1976; Dott et al., 1976). Growth of bacteria with similar

activities must be prevented. Acetaldehyde production by

contaminating microflora can be minimised by using sound, clean

fruit. Products where possible should be kept in anaerobic

conditions and at a low pH value to minimise oxidation of sulphite

and to maximise the concentration of active molecular SO^. Improved

factory hygiene and a rigid sanitation programme for the processing

of equipment help to minimise the presence of potential sulphite-

binding compounds. Sulphur dioxide treatment concentrations must be

calculated to give optimal effect according to the pH value and

content of sulphite-binding compounds (Beech et al. , 1979).

Hazards of Using Sulphiting Agents

Recently the continued large-scale use of SO^ has been brought

into question for more serious reasons. The Acceptable Daily Intake

(ADI) for sulphites set by the Life Science Research Office in 1985

is 42 mg for a 60 kg person. It is estimated that the total, intake

of sulphites is about 10 mg per person every day although it is not known what proportion of this is in the free molecular form of SO^.

Sulphiting agents are categorised as being Generally Recognised as

Safe (GRAS) provided they are not used in meats or other foods

recognised as a dietary source of thiamin. However, this GRAS

status is presently under review in the light of continuing reports

of toxicity apparently caused by SO^.

The relative toxicity of the free and bound forms of SO^ is

still not known but, by virtue of their relative stabilities, it is

thought likely that free SO^ poses the greater hazard. Numerous

cases of sulphite-induced asthma attacks have been reported in

medical literature since 1977 (Baker et al., 1981; Bush et al.,

1986). Many of these cases were confirmed with positive challenges

with capsules or solutions containing inorganic sulphite.

Free sulphite is metabolised principally by sulphite oxidase

producing sulphate which is safely excreted in urine. Normally

individuals have a tremendous capacity to metabolise sulphite.

Profound sulphite oxidase deficiency has been recorded in a very

few fatal cases and is characterised by increased urinary excretion

of sulphite. Alarm at the widespread usage of SO^ was heightened by

suggestions of its mutagenic effects reported by Mukai et al.

(1970) who reported mutagenesis of E. coli after exposure to sodium

bisulphite, but there is no evidence of mutagenesis caused by

sulphites in human cells.

Although asthmatic reactions continue to be the most common

adverse reaction, individuals have also experienced urticaria,

pruritis and swelling of the tongue, while oral challenges produced

nausea, flushing and erythema sometimes causing hypertension and

anaphylactic-like reactions (Green, 1976; Prenner and Stevens,

1976; Taylor et al., 1986).

Thankfully these rather alarming adverse reactions are

relatively uncommon but are certainly undesirable. Pressure is

being brought to bear upon manufacturers to lower the permitted

levels of sulphite in their products. Unfortunately there is no

suitable substitute for sulphiting agents as they have so many

desirable properties, but the need for SO^ can be decreased by

minimising contamination, avoiding oxidation, using optimum

sulphite concentrations and keeping the pH value as low as

possible. Wherever possible formation of sulphite-binding compounds

should be prevented and SO^ conserved by packing products under

anaerobic conditions. As Erik Millstone (1985) wrote "Risks which

arise from the use of additives are borne almost entirely by the

consumer" and he points out that additives are used by industry

when their use serves the economic interest of industry. When put

in this light it becomes obvious why we must regulate and monitor

the use of additives and question the advantages and more

importantly the disadvantages of their inclusion in our daily diet.

YEASTS AND FOOD SPOILAGE

Spoilage YeastsProducts affected by food-spoilage yeasts are generally acidic

(pH 2.5 - 4.5) and may contain high concentrations of sugars,

ethanol or carbon dioxide. Such yeasts are not known to be toxic or

produce serious off flavours, but spoil the product either by

producing carbon dioxide causing distortion or explosion of

packaging, or by giving a visible haze or sediment which are

unacceptable in wines and clear drinks. A list of commonly isolated

spoilage yeasts that contaminate preserved acid foods include:

Zygosaccharomyces bailii, Zygosaccharomyces bisporus,

Zygosaccharomyces rouxii, Pichia membranaefaciens, Candida krusei,

Brettanomyces spp. , Torulopsis spp. and Schizosaccharomyces pombe

(Warth, 1986). Rehm and Wittman (1962) determined inactivation

concentrations of SO^ for a variety of yeast species finding

strains of Saccharomyces and Zygosaccharomyces tolerant to

concentrations of SO^ ranging between 0.10 - 20.20 ppm and 7.2 - 8.7 ppm, respectively. Dott and Truper (1978) found "killer

yeasts" which were highly resistant to SO^ and which, when grown in

mixed cultures, cause death of other yeasts by producing sulphite.

Warth (1986) reviewed the relative sensitivities of a number of

yeast strains to SO^, benzoic acid and sorbic acid and found that,

generally, a strain tends to be resistant to all three acid

preservatives or none (Table 3). He suggested that all three

preservatives may have a common mechanism of action. In a previous

publication, Warth (1985) highlighted the considerable range of

tolerances to sulphite among yeast strains. Kloeckera apiculata, a

yeast found in the early stages of spontaneous fermentation of

grape musts (Kunkee and Goswell, 1977) and apple juices (Beech and

Carr, 1977), is much more sensitive to sulphite than strains of

Zygosacch. bailii which is generally regarded as a resistant strain.

Table 3. Maximum concentrations of preservative tested permitting

anaerobic growth of yeasts at pH 3.5. Reproduced from

Warth (1985).

Species Sorbic acid

(mM)

Benzoic acid

(mM)

Free SO^

(mM)

Kloekera apiculata 1 1.5 0.05

Saccharomyces cerevisiae 1297 1 0.7 < 0.14

Saccharomyces cerevisiae 1298 2 2 0.51

Candida krusei 3 3 0.48

Saccharomycodes ludwigii 3 3 2.2Schizosaccharomyces pombe 4 4 1.9

Zygosaccharomyces bailii 2476 2 2 2.8Zygosaccharomyces bailii 1292 4 4 2.6Zygosaccharomyces bailii 2227 4 4 2.8

Spoilage yeasts were seen to tolerate a considerable range of

concentrations of SO^ (Balatsouras and Polymenacos, 1963) and

Zygosacch. bailii consistently appears as a troublesome food

spoiler (Pitt and Richardson, 1973; Rankine and Pilone, 1973;

Thomas and Davenport, 1985).

Mechanisms of Action of Sulphur Dioxide on YeastsThe mechanism of the antimicrobial action of SO^ is known to be

complex, with possible targets in the cell wall, plasma membrane

and dispersed throughout the cytoplasm. As the susceptibility of

any organism depends upon exposure of target sites to the

preservative, it is essential to understand the kinetics of SO^

transport into the cell. Sulphite may be taken up by an active or

passive system which is believed to differ among micro-organisms.

Any explanation must take into consideration the molecular

composition, organisation and function of the plasma membrane since

all of these factors are likely to influence solute transport.

Sulphur Dioxide Transport

Although there has been widespread study of sulphate transport

in yeasts (Horak et al., 1981; Benitez et al., 1983; Garcia et al.,

1983; Alonso et al., 1984), there is relatively little published

material specifically related to sulphite or sulphur dioxide

transport. McCready and Din (1974) were the first to propose an

active transport system for sulphate in Sacch. cerevisiae which was

confirmed in 1977 by Breton and Surdin-Kerjan who found a biphasic

transport system involving two distinct permeases. However, the

currently accepted mechanism of transport of sulphite into Sacch.

cerevisiae and S * codes ludwigii is that of free diffusion of the

molecular form of SO^ (Stratford and Rose, 1986; Stratford et al.,

1987) which conflicts with the active transport system previously

proposed by Macris and Markakis (1974). Stratford and Rose (1986)

presented strong evidence in favour of a protein not being involved

in SO^ transport in the form of near vertical Woolf-Hofstee plots

(referred to in this thesis as Woolf-Eadie plots) at pH 3.0 and 4.0

(Hofstee, 1959). Values for calculated from kinetic plots of v

against v/s were 3.2 mM and 0.1 mM at pH 3.0 and 4.0, respectively,

where v is the initial velocity of sulphite accumulation and s the

extracellular SO^ concentration. These values are far in excess

of the concentration of SO^ required to kill Sacch. cerevisiae

suggesting that passive transport predominates under these

conditions. This evidence is supported by the inability of

carbonylcyanide m-chlorophenylhydrozone (CCCP) and dinitrophenol

(DNP) (Borst-Pauwels, 1981) to affect initial velocities of

sulphite accumulation. These protonophores are known to dissipate

the transmembraneous proton gradient (apH) and to inhibit mediated

transport systems. Further evidence for the lack of active

transport of SO^ came from the finding that exclusion of glucose

from the reaction mixture had no effect on initial velocities of

accumulation. Similarly, inability of the glycolytic inhibitor

2-deoxyglucose to affect SO^ uptake adds fuel to the theory that energy is not required for SO^ accumulation. Additional evidence is

provided by the absence of an effect of pH value on the process,

atypical of protein-mediated transport.

Macris and Markakis (1974) studied the kinetics of

radiolabelled SO^ uptake by Sacch. cerevisiae var. ellipsoideus

making some valuable observations on SO^ toxicity and pH

dependence. There is a close correlation between accumulation of 35radiolabel from [ S]sulphite, over the pH range 3.0 - 5.0, and

concentration of S0^ in solution, which is corroborated by Hinze

and Holzer (1985a) and Stratford and Rose (1986). Evidence strongly

suggests that, over this pH range, only the molecular form of SO^

passes into organisms and by inference that Sacch. cerevisiae do

not transport sulphite (HSO^ ). In these organisms, plasma

membranes merely act as selective barriers to free diffusion of

SO^. For this reason, the relative structure and fluidity of the

plasma membrane most probably affect solute transport, and further

investigations are necessary in this area. This aspect will be

covered more thoroughly in the following sections.

A slow transport system for HSO^ in Sacch. cerevisiae has been

tentatively suggested which is evident in the presence of low

concentrations of molecular SO^ (Stratford and Rose, 1986). As

sulphite concentrations are increased, this system rapidly becomes

saturated and masked by diffusion of higher concentrations of

molecular SO .

Intracellular Effects of Sulphur Dioxide

Saccharomyces cerevisiae and S * codes ludwigii accumulate SO^

initially very rapidly reaching a plateau concentration after about

five minutes exposure. Intracellular SO^ concentrations at

equilibrium are many times greater than in suspension (Stratford

et al., 1987). This can be explained by the dynamic equilibrium

between the three forms of sulphur dioxide, sulphite and bisulphite

in solution and the presence of sulphite-binding compounds

(Burroughs and Sparks, 1964a). Intracellular pH values in Sacch.

cerevisiae lie in the region of pH 6.5 where only 0.0015% of free

sulphite exists in the molecular form (King et al., 1981). If the

extracellular pH value is below pH 6.5, molecular SO^ will

accumulate and dissociate inside the cell until concentrations of

SO^ are equal on both sides of the plasma membrane resulting in

acidification of the cytoplasm. It is conceivable that there may be

leakage or active expulsion of anions from the cell resulting in a

net flow of protons into the cell which will either equilibrate the

cytoplasmic pH value with that of the medium or impose a heavy

energy load on the cell in expelling protons. The extent of SO^

accumulation must depend upon the intracellular pH value in the

organisms, so differences in resistance between organisms may be

attributed to differences in intracellular pH value or, by

implication, their ability to maintain constant intracellular pH

values (Sigler et al., 1981a, b; Salmond et al., 1984).

The antimicrobial activity of lipophilic acid food

preservatives has been attributed to inhibition of transport

mechanisms by lowering the A pH component of the proton-motive force

(Freese et al., 1973). Salmond et al. (1984) studied the effect of

weak acid preservatives on E. coli and concluded that, although

accumulation of acid in the cells resulted in a decrease in the

intracellular pH value, this was not the primary cause of growth

inhibition. It was significant that these workers found the

intracellular pH value of organisms was lowered to a greater extent

by food preservatives than by weak acids with a similar pK value.

They suggested that the inhibitory effect of unidentified metabolic

functions by the undissociated acid had a synergistic effect with

accumulation of the acid on intracellular pH values. It was

suggested by Stratford et al. (1987) that the relative resistance

of S' codes ludwigii may at least be partially attributed to its

increased capacity to produce sulphite-binding compounds,

specifically acetaldehyde and to a lesser extent pyruvate, compared

with Sacch, cerevisiae, and to its decreased capacity to accumulate

SO^. Stratford et al., (1987) also postulate that S'codes ludwigii,

having a plasma membrane richer in C phospholipid fatty-acyllo ! 1residues compared with Sacch. cerevisiae, may have a more fluid

membrane thereby facilitating diffusion of SO , a theory that will

be discussed more fully later in this Introduction.

Sulphur Dioxide Targets

Sulphite will react with a wide variety of cell constituents as

suggested earlier and, by implication, is likely to influence the

cell at a number of target sites. Outside the cell, SO^ binds with

many compounds rendering them unavailable for yeast nutrition.

Portnova (1978) demonstrated that an increase in the concentration

of SO^ added to grape must, from zero to 282 ppm, resulted in a

decrease in the lipid content of yeasts, particularly in lipids

containing unsaturated fatty-acyl residues. When the SO^

concentration was increased from 192 to 282 ppm it also caused a

decrease in the lipid content of wine particularly in the amount of

unsaturated fatty acids present essential to the anaerobic growth

of certain yeasts (Andreason and Stier, 1954).

Anacleto and van Uden (1982) proposed that SO^ acts upon a

yeast cell in three stages. Firstly, SO^ binds to receptors on the

cell surface. Next, membrane damage occurs due to a change in

activity of the receptor-sulphur dioxide complex. Thirdly, the cell

loses viability. Two distinct receptors for SO^ in Sacch.

cerevisiae are proposed. One is the "sulphur dioxide death site", a

membrane protein with a high affinity for SO^ exposed to the outer

surface of the plasma membrane. Combination of this protein with

SO^ causes a lowering of the free energy of activation of the

denaturing process resulting in loss of viability. The second

receptor is thought to modulate the entropy of activation of the

"death site". These workers suggested that the first receptor may

be the same target proposed by Schimz and Holzer (1979), and that

the receptor was membrane-bound ATPase which, when bound to sulphur

dioxide, hydrolyses intracellular ATP in an uncontrolled way,

depleting intracellular ATP. However, Hinze and Holzer published

data (1985b) showing how concentrations of SO^ up to 0.5 - 5.0 mM

lead to depletion of cellular ATP mainly as a result of

inactivation of glyceraldehyde 3-phosphate dehydrogenase, an enzyme

intimately involved in degradation of carbohydrates yielding ATP.

At the same time a 10 to 100 fold increase in concentration of

glyceraldehyde 3-phosphate over the concentration found in the

absence of sulphite was observed. This gross depletion of ATP

caused by sulphite is probably the major cause of cell death

(Schimz, 1980). Prior to cell death, the rapid decrease in the

cellular content of ATP was accompanied by an increase in the level

of inorganic phosphate while the content of ADP remained reasonably

constant (Schimz and Holzer, 1979; Schimz, 1980). Concentrations of

other ribonucleoside di- and triphosphates in sulphite-treated

cells showed parallel changes to ATP. In addition, Schimz and

Holzer (1977) showed that low sulphite concentrations inhibited the

viability of yeast populations.

The extent of the damage imposed on organisms is dependent upon

the concentration of sulphite, pH value, physiological condition,

density and age of organisms, and on incubation time. If the yeast

population was exposed to sulphite for less than one hour, the

lethal effect could be prevented and depletion of cellular ATP was

reversible. Cultures treated with a sub-lethal dose of SO^

characteristically showed increased lag times, up to 600 h (Warth,

1985) but, when growth occurred, there was no decrease in growth

rate or final yield. In 1986 Hinze and Holzer demonstrated that

inhibition of ATP production by SO^ is confined to inhibition of

substrate-level phosphorylation at the level of glyceraldehyde

3-phosphate dehydrogenase and not respiratory-chain

phosphorylation. This was confirmed by revealing the same rate of

ATP decrease in respiratory-deficient mutants (pet 936), which lack

mitochondrial F^ATPase, as in the wild-type strain of Sacch.

cerevisiae X2180. However, in vitro experiments with purified

ATPase from yeast mitochondria revealed a sensitivity of this

enzyme to sulphite (Maier et al., 1986). Maier et al. (1986)

therefore propose that sulphite acts both on glycolysis and on

respiratory-chain phosphorylation. Both oxygen consumption and the

ATP content of glucose-starved yeast were drastically lowered by

sulphite during incubation at pH 3.6. Sulphite may impair

respiration by reacting with flavoproteins; for example, cytochrome

b (1-lactate dehydrogenase) is known to be competitively inhibited•—2by sulphite (Lederer, 1978).

It is possible that these critical targets in organisms may

vary in their sensitivity to SO^, or simply that the physical

exclusion of SO , brought about by variable rates of SO^ uptake or

the mopping up of free SO^ by binding compounds, will impart a

relative degree of resistance to an organism.

In addition to inactivation of glyceraldehyde 3-phosphate

dehydrogenase, formation of an acetaldehyde-bisulphite complex with

glyceraldehyde 3-phosphate, which slows down the rate of the

dehydrogenation by lowering substrate concentration, may also

contribute to depletion of ATP. Sulphite also binds glucose and

dihydroxyacetone phosphate thereby inhibiting operation of the

Embden-Meyerhof-Parnas pathway (Beech and Thomas, 1985). Any

activity of the TCA cycle is also decreased since sulphite binds

oxaloacetate and glutaric acid, and this may account for the drop

in oxygen consumption by sulphited cells (Rehm, 1964). Nicotinamide

adenine dinucleotide itself reacts with SO^ (Johnson and Smith,

1976; Tuazon and Johnson, 1977), and Rehm (1964) has shown that

NAD+-dependent steps of glycolysis in Sacch. cerevisiae were

strongly inhibited by sulphite. As a result of sulphite-induced

depletion of the intracellular ATP pool and inhibition of ATP

production, many ATP-dependent processes are halted, e.g. the

sulphite permease (Kleinzeller et al., 1959) and ATP sulphurylase

(de Vito and Dreyfuss, 1964).

Intracellular effects are not confined to inhibition of

metabolic pathways. Structural damage may also occur due to

distortion of structural proteins or peroxidation of membrane

lipids (Utsumi et al., 1973).

Stratford (1983) examined the effect of sulphite on initial

velocities of accumulation of the amino acids arginine and lysine

and of glucose by Sacch. cerevisiae NCYC 366. Accumulation of both

amino acids was inhibited after addition of sulphite (0.5 mM) to a

cell suspension containing the amino acid (1 - 10 mM), glucose

(100 mM) and organisms (0.5 mg dry wt ml ), but sulphite did not

affect the rate of accumulation of glucose. It was concluded that

sulphite had caused a dissipation of the proton-motive force that

is created across the plasma membrane, thereby inhibiting active

transport of solutes. Alternatively, sulphite might cause

denaturation of transport proteins exposed on the outer surface of

the plasma membrane.

Stimulation of Production of Sulphite-Binding Compounds

The SO^ resistance of spoilage yeasts has partly been

attributed to the variable ability of yeasts to produce

sulphite-binding compounds, particularly acetaldehyde, that bind

sulphite to form a-hydroxysulphonates. This is especially so when

strains are grown in the presence of sulphite (Rankine, 1968;

Weeks, 1969), so rendering free SO^ ineffective (Rankine and

Pocock, 1969; Stratford et al., 1987). This ability of SO^ to

stimulate acetaldehyde production has long been recognised as

Neuberg's second form of yeast fermentation (Neuberg and Reinfurth,

1918, 1919) resulting in net accumulation of glycerol, compared

with Neuberg's first form of fermentation which leads to production

of ethanol. Freeman and Donald (1957) summarised Neuberg's second

form of fermentation as follows:

C6H12°6 + NaHS03 ^ CH3CH0.NaHS03 + C H 0 + C02Glucose Bisulphite Acetaldehyde- Glycerol

bisulphite

During the course of a normal fermentation NADH, formed during

oxidation of 3-phosphoglyceraldehyde to 3-phosphoglyceric acid, is

re-oxidized when acetaldehyde is reduced to ethanol. In the

presence of sulphiting agents, acetaldehyde becomes bound and can

no longer serve as the hydrogen acceptor for NADH. Under these

conditions, dihydroxyacetone phosphate becomes a substitute

hydrogen acceptor for NADH resulting in formation of glycerol

3-phosphate and subsequent accumulation of glycerol (Nord and

Weiss, 1958). The steering action of sulphite has been exploited in

production of glycerol, notably during World War I where

approximately 1,000 tons of glycerol per month were manufactured by the "sulphite process" (Lawrie, 1928). The process was

comprehensively reviewed in following years (Prescott and Dunn,

1949; Underkofler, 1954), but there are very little data available

in recent publications. Yields of glycerol were found to depend on

concentration and type of carbohydrate substrate, concentration of

sulphite, yeast strain and size of inocula, surface volume ratio,

pH value and temperature (Lees, 1944; Wright et al., 1957; Kalle

and Naik, 1985).

Although acetaldehyde is recognised as the primary sulphite-

binding compound, pyruvic acid and 2-oxoglutaric acid are known to have significant binding capacities (Rankine and Pocock, 1969;

Weeks, 1969). During the fermentation of three grape juices by

eight yeasts (Sacch. spp.), these constituents resulted in 49 - 83%

of measured sulphite being bound. The maximum range of

concentrations of the binding components for individual wines were

10 - 48 ppm for acetaldehyde, 9 - 7 7 ppm for pyruvic acid and 5 -

63 ppm for 2-oxoglutaric acid, depending on the yeast strain and

nature of the grape juice. The amount of acetaldehyde produced was

directly related to the total SO^ present, and both of these

factors were related to the strain of yeast used. When a subsequent

addition of SO^ was made after fermentation was complete, the

amount bound depended largely on the concentrations of pyruvic and

2-oxoglutaric acids present (Rankine and Pocock, 1969).

It is not clear from these investigations whether production of

pyruvate and 2-oxoglutarate is actively stimulated by SO^. Weeks

(1969) reports that pyruvate concentrations are increased in the

presence of SO^, and this has been corroborated more recently by

Stratford et al. (1987) who recorded production of pyruvate by

Sacch. cerevisiae TC8 reaching 20 - 40% of the concentration of acetaldehyde in the presence of sulphite. In cultures of S1 codes ludwigii, however, there were negligible concentrations of

pyruvate.

Resistance to Sulphur Dioxide

Tolerance of yeasts to sulphur dioxide falls into two

categories, namely inherent tolerance and inducible tolerance.

Inherent tolerance of strains like Zygosacch. bailii and S * codes

ludwigii (Ingram, 1960; Reed and Peppier, 1973) is genetically

determined (Zambonelli et al., 1972) and transmitted to subsequent

generations even under sulphite-free conditions. Opinions vary

regarding the ability of yeasts to acquire SO^ resistance. Beech

and Thomas (1985) showed that a resistant strain of Zygosacch.

bailii, if left to acclimatise for 14 days in media containing 3 mg

molecular SO^ 1 \ eventually grew even though the concentration of

SO^ when growth occurred exceeded that normally expected to prevent

growth. These workers postulated that the organisms had acquired

resistance.

The nature of inherent SO^ resistance may be a reflection of

different target sites in different species, for example, in the

conformation of the "sulphur death site" receptor or in the rate of

uptake of SO^. In addition, yeasts can detoxify SO^. Sulphite

reductase, which has been detected in yeasts, converts SO^ to

sulphide (Wainwright, 1967) and has an integral role in sulphate

metabolism in yeasts and may be involved in SO^ resistance.

Intracellularly, sulphate is converted to adenosine

5'-phosphosulphate which is then converted to the high-energy

intermediate 3'-phosphoadenosine 5'-phosphosulphate (PAPS; Robbins

and Lipman, 1958); PAPS is then reduced to sulphite which is

finally reduced by sulphite reductase to sulphide (Yoshimoto and

Sato, 1968a, b, 1970; Prabhakararao and Nicholas, 1969, 1970).

Warth (1977) proposed that the resistance of Zygosacch. bailii

to acid preservatives, including sorbic and benzoic acids and SO^,

was primarily from the activity of an inducible energy-requiring

pump that transports preservative molecules out of the cell. This

explained the enhanced resistance of organisms grown at high

concentrations of glucose (Pitt, 1974) in terms of the high energy

demands of this resistance mechanism. Support for this theory is

lacking as a mechanism of resistance, because of inability to

demonstrate a specific pump and considering the insurmountable task

of ejecting the rapidly penetrating acid (Macris, 1975; Cole and

Keenan, 1987). Cole and Keenan (1987) investigated the effect of

benzoic acid on Zygosacch. bailii NCYC 563 and propose that, by

decreasing the protoplast volume and concentrating cellular

components, the buffering capacity of organisms may be increased.

At the same time, these organisms were able to increase acid efflux

either by proton extrusion directly through the plasma membrane

ATPase (Peters and Borst-Pauwels, 1979; Serrano, 1980) or by

excreting organic acids produced during normal metabolism that do

not rapidly re-enter cells (Sigler et al., 1981b; Opekarov^ and

Sigler, 1982).

YEAST PLASMA MEMBRANE: COMPOSITION AND FUNCTION

The yeast plasma membrane has several important functions.

Firstly, it acts as a protective barrier enabling the maintenance

of a constant internal environment inside the cell. Secondly, by

selectively controlling the passage of solutes and metabolites, it

allows interaction with the extracellular medium. Finally it serves

as an organelle on which enzymic reactions leading to synthesis of

wall components may occur.

In general yeast plasma membranes contain, in terms of dry

weight, approximately 40% lipid and 60% protein held together by

non-covalent interactions. The proportions tend to vary between

reports and organisms, largely because of differences in

experimental technique (Rank and Robertson, 1983). Some

carbohydrate is also usually present covalently linked to lipid or

protein and in the hydrated state, comprising approximately 20% water which is tightly bound and essential for maintenance of

structural integrity (Harrison and Lunt, 1980).

Data related to the composition of the plasma membrane in

Sacch. cerevisiae are limited and those related to the organelle in

Zygosacch. bailii are even more scarce. Detailed analyses of plasma

membranes of a strain of Sacch. cerevisiae were first obtained by

Longley et al. (1968). The membranes were obtained by osmotic lysis

of yeast spheroplasts, and the analyses confirmed in 1971 by Hunter

and Rose. About 50% of the dry weight of the membrane was accounted

for by protein and approximately 40 - 45% by lipid (Boulton, 1965; et s!->

Longley^ 1968; Schibeci et al. , 1973), with the remainder probably

being carbohydrate.

Although proteins comprise a significant proportion of the

plasma membrane in yeasts they have not been fully characterised to

date. Perhaps the most extensive contribution to analysis of

plasma-membrane proteins of Sacch. cerevisiae has been made by

Santos and his colleagues (Santos et al., 1978, 1982). They

detected 25 polypeptides and 12 glycoproteins with molecular

weights between 10,000 and 300,000 when proteins isolated from

plasma membrane of Sacch. cerevisiae were analysed by

one-dimensional sodium dodecyl sulphate-polyacrylamide gel

electrophoresis (SDS-PAGE). High molecular-weight proteins were

predominant. A similar diversity of polypeptides was observed by

Schneider et al. (1979) who isolated 17 - 19 predominantly high

molecular-weight proteins from plasma-membrane preparations of

Candida tropicalis. Some individual yeast plasma-membrane proteins

have been studied. An example is the general amino-acid permease

(GAP) which catalyses the uptake of a wide variety of amino-acids

in Sacch. cerevisiae (Woodward and Kornberg, 1980).

The lipid fraction, which is fairly well characterised in the

plasma membrane of Sacch. cerevisiae, can be divided into two main

classes, namely polar and neutral lipids. Polar lipids in

eukaryotic micro-organisms are principally amphipathic glycero-

phospholipids, glycolipids and free sterols; neutral lipids

comprise triacylglycerols and sterol esters. There are considerable

discrepancies in the published literature concerning the relative

proportions of each lipid class present in the plasma membranes of

Sacch. cerevisiae. Kramer et al. (1978) reported that plasma-

membrane phospholipids of Sacch. cerevisiae comprised only 5 - 6 %

of the total cellular lipid compared to Kaneko et al. (1976) who

found that phospholipids constitute over 50% of the total cellular

lipid of Sacch. cerevisiae infering a high plasma-membrane

phospholipid content. Arnold (1981) surmised that the low values

obtained by Kaneko et al. (1976) and Schneider et al. (1979) were

artefactual arising from enzymic degradation of phospholipids by

non-specific lipase and phospholipases, since both groups of

workers, in a similar study on C. tropicalis, reported an

abnormally high content of free fatty acids in their

plasma-membrane preparations. Rattray (1988), in a general review,

reports that cellular phospholipids in 18 different strains of

Sacch. cerevisiae contribute between 17 and 66% of the total lipid fraction. This compared with the one strain of Zygosacch. bailii

reported in which the total lipid fraction comprised approximately

15% phospholipid (Malkhas'Yan et al., 1983). Nurminen et al. (1976)

reported that Sacch. cerevisiae, grown under glucose-repressed

conditions, had over 80% of the total cellular phospholipid and

sterol in the plasma-membrane fraction.

Rank and Robertson (1983) reported the relative proportions of

lipid classes in yeast plasma-membrane vesicles that were

aggregated to remove non-plasma-membrane vesicles. They contained

45% phospholipids, 21% free fatty acids, 16% sterols, 8% sterol esters, 5% tri-acylglycerols and 5% di-acylglycerols, compared with

non-aggregated vesicles containing 9% phospholipids, 67% free fatty

acids, 20% sterols and minor quantities of tri- and di-acylgly­cerols and sterol esters. The high concentrations of free fatty

acids were again attributed to lipase activity, while phospholipase

activity was thought to result in lowering measurable

concentrations of phospholipid by the formation of glyceropho-

sphorylcholine from phosphatidylcholine.

With improved purification techniques it seems likely that

further studies will show that phospholipids and free sterols

constitute the major portion of plasma-membrane lipid in Sacch.

cerevisiae, as is the case in plasma membranes derived from other

eukaryotic organisms (Harrison and Lunt, 1980). Neutral sterol

esters and triacylglycerols usually account for most of the

remaining plasma-membrane lipid with minor quantities of free fatty

acid and mono- and di-acylglycerols (Rattray, 1988).

Glycerolphospholipid is a general term applied to any lipid

containing phosphoric acid as a mono- or di-ester, in which a

hydrophilic head-group is linked via a glycerol residue to a

hydrophobic tail consisting of two long-chain fatty-acyl residues

esterified to hydroxyl groups of the glycerol moiety. Both the

chain length and degree of unsaturation vary in the hydrophobic

tail region. Aerobically-grown Sacch. cerevisiae was found to

contain and residues constituting between 70 and 80% of

the total fatty-acyl residues present in plasma-membrane

preparations (Longley et al., 1968; Schneider et al., 1979).

Cartwright (1986) and Cartwright et al. (1987) found that the

relative proportions of fatty-acyl residues within the

plasma-membrane phospholipids of Sacch. cerevisiae were similar to

those reported by Beavan et al. (1982) for whole-cell

phospholipids. The phospholipids from Zygosacch. bailii

characteristically contain predominantly C and C fatty-acyllo11 loI 2residues which constitute approximately 75% of total phospholipids

(Viljoen et al., 1986).

The composition of the hydrophilic head group is also variable,

but shows a similar composition in most yeasts. Chemical structures

of the four major classes of phospholipid found in yeast are shown

in Figure 1. Generally phosphatidylcholine (PC) and phosphatidyl-

ethanolamine (PE) predominate comprising between 20 and 50%, and 15

and 40% of total cellular phospholipids respectively, with 10 to

15% phosphatidylinositol (PI) and 5 to 15% phosphatidylserine (PS)

(Longley et al., 1968; Rank et al., 1978; Rattray, 1988).

The presence of other minor classes of phospholipid (less than

20% of total phospholipids) has been reported including phosphatidylmonomethylethanolamine (PMME), phosphatidyl-

34.

dimethylethanolamine (PDME), phosphatidic acid (PA),

lysophosphatidylethanolamine (LPE), diphosphatidylglycerol (DPG)

and phosphatidylglycerol (PG) (Letters, 1966; Getz et al., 1970;

Steiner and Lester, 1972b). It is, however, generally accepted that

many of these minor components arise by uncontrolled action of

phospholipases during lipid extraction (Ratledge and Evans, 1987).

Henry (1982) found the proportions of phospholipid classes found in

plasma membranes mirror those found in the whole cell.

Although the relative proportions of phospholipid classes is

relatively constant in yeasts, it is significant that

phosphatidylserine and phosphatidylinositol are conspicuous by

their lack of unsaturated fatty-acyl residues compared to the other

phospholipids (Rattray et al., 1975; Watson and Rose, 1980).

Sterols have a fused cyclopentanoperhydrophenathrene ring

system forming a rigid backbone with eight to ten carbon atoms in a

side chain at C-17 and a hydroxyl head group at C-3. The hydroxyl

group represents the polar moiety while the non-polar side chain

and steroid skeleton constitute the hydrophobic region of the

molecule. Ergosterol is the major sterol component of yeast plasma

membranes (Nurminen et al., 1975) and of whole cells (Dulaney

et al., 1954; Nes et al., 1978) representing 0.03 to 4.6% of yeasts

on a total dry weight basis (El-Refai and El-Kady, 1968). The

second most common sterol is the precursor of ergosterol,

24(28)-dehydroergosterol, found by Longley et al. (1968) to appear

in roughly equal proportions to ergosterol in Sacch. cerevisiae

NCYC 366. Small amounts of zymosterol have also been found in many

yeasts (Dulaney et al., 1954; Hossack et al., 1977a; Marriot,

1975).

Figure 1. Chemical structures and space-filling atomic models of

(a) phosphatidylethanolamine, (b) phosphatidylcholine,

(c) phosphatidylserine and (d) phosphatidylinositol.

Carbon atoms are indicated in black, hydrogen atoms in

white, oxygen atoms are dotted, double dotted with double

bonds, nitrogen atoms are also dotted and phosphorus

atoms are striped.

Figure 1 .35.

c-o c-oc-o c-oc-o c-oc-o c-o

CH-CH;

CHaCH-CHa

CHa

O - P - O

' >-OH OH J-OM

HO

H<yNHa

•o

The lipid composition of yeasts is very sensitive to changes in

the extracellular environment (Hunter and Rose, 1971; Rattray

et al., 1975). Both physical and chemical factors are important

including growth rate, composition of medium, temperature and

dissolved oxygen tension (Jollow £t al., 1968; Hunter and Rose,

1972; Prasad, 1985). Oxygen has a pronounced effect on the growth,

general metabolism and lipid composition of yeasts resulting in

specific changes in plasma-membrane composition. This finding has

been exploited in a technique developed by Alterthum and Rose

(1973). Andreasen and Stier (1953, 1954) discovered that Sacch.

cerevisiae has a nutritional requirement for a sterol and an

unsaturated fatty acid when grown anaerobically. These compounds

cannot be synthesised anaerobically because the fatty acid

desaturase enzyme and enzymes involved in the conversion of

squalene to ergosterol require molecular oxygen. Other

quantitatively minor anaerobically-induced requirements such as

nicotinic acid (Suomalainen et al., 1965) are usually supplied by

low concentrations of yeast extract (Alterthum and Rose, 1973).

Although it is generally believed that the requirements for an

unsaturated fatty acid are fairly non-specific (Light et al., 1962)

there is evidence that the same is not true for sterols (Nes

et al., 1976, 1978; Pinto and Nes, 1983). These workers were able

to show that, by comparing pairs of sterols differing in only one

component, each structural feature of ergosterol appeared to have

some functional significance in the yeast, and the ability of

different sterols to support anaerobic growth is not simply an all

or nothing phenomenon as had previously been implied (Proudlock

et al., 1968; Hossack and Rose, 1976). The natural yeast sterol,

ergosterol, was the most capable of supporting anaerobic growth.

This anaerobic auxotrophy has been exploited by many workers to

change the lipid composition of the plasma membrane, particularly

the degree of fatty-acyl saturation, to probe basic relationships

between composition and function in plasma membranes from Sacch.

cerevisiae (Thomas et al., 1978; Thomas and Rose, 1979; Keenan

et al., 1982; Calderbank et al., 1984, 1985). The supplemented

fatty acid has been shown to account for between 50 and 69% of the

residues within the phospholipids, and between 47 and 92% of those

in triacylglycerols (Watson and Rose, 1980), depending on the

unsaturated fatty acid supplement and strain of Sacch. cerevisiae.

The proportions of phospholipid classes can also be affected by

specific supplements (Hossack et al., 1977b). Under aerobic

conditions, low concentrations of choline in a chemically defined

growth medium induced Sacch cerevisiae to synthesise a greater

proportion of phosphatidylcholine resulting in a three-fold

increase in this phospholipid (Waechter et al., 1969; Waechter and

Lester, 1971). Similarly, phosphatidylethanolamine synthesis could

be increased two-fold with the inclusion of ethanolamine in the

growth medium (Ratcliffe et al., 1973). Buttke et al. (1982),

however, found they were able to modulate the fatty acid

composition of phosphatidylethanolamine independently of the other

phospholipids in a mutant strain of Sacch. cerevisiae by exploiting

the preference to incorporate unsaturated fatty acids into

phosphatidylethanolamine. The phospholipid fatty-acyl composition

could also be altered in response to different sterols (Wieslander

et al., 1981; Buttke et al., 1982). Mutant strains have also been

employed to explore the relationship between membrane fluidity,

composition and cell growth (Barber and Lands, 1973; Holub and

Lands, 1975; Esfahani et al., 1981a).

Structure of the Plasma MembraneDanielli and Davson (1935) were among the first to propose a

realistic model describing membrane structure and composition. They

envisaged a phospholipid bilayer held together by van der Waals

forces with the polar head groups aligning on the outer surfaces

and the hydrophobic tails of the lipid molecules sandwiched inside

the membrane. Proteins were thought to be spread on the surface of

the polar head groups, but, at that stage, their role was not

understood. Subsequently, additional information was building up

about the roles of proteins, and it gradually became clear that

proteins are partially or completely embedded on each side of the

membrane. This led to the development of more flexible model

systems, including the lipoprotein sub-unit model (Lucy and

Glauert, 1964), the mosaic model (Lenard and Singer, 1966) and

culminated in the suggestion of Singer and Nicolson (1972). Today

the Singer and Nicolson (1972) model is regarded as a grossly

simplistic and inadequate model but still forms the basis of modern

membrane models. It describes a bilayer consisting of oriented

lipid molecules similar to the Davson model in which two types of

protein are embedded. Firstly, extrinsic proteins, like cytochrome

c that are water soluble but function when bound to the membrane

surface, are loosely attached to lipid headgroups or other membrane

proteins by ionic or hydrogen bonds; secondly, intrinsic

amphipathic globular proteins which are tightly bound and

incorporated to various degrees into the fluid lipid bilayer. The

essential features of this model are that membranes can exhibit an

asymmetric distribution of proteins and lipids, and that lipids in

the bilayer exist predominantly in a fluid state. This makes some

provision for lateral and rotational movements of lipids and

proteins, so that selective exchange of hydrophilic compounds can

occur, and from a thermodynamic point of view maximising

hydrophobic and hydrophilic interactions. However, the model has

subsequently been criticised as it leaves the impression that the

only function of membrane lipids is to provide a hospitable

environment of proper fluidity and makes no provision for

lipid-lipid, protein-lipid (Chapman et al., 1979) and

protein-protein interactions which may be important in influencing

membrane fluidity and intrinsic protein conformation (Boggs, 1980).

The presence of intrinsic proteins has been shown to affect the

conformation of neighbouring lipids (Jost et al., 1973) and the

effects of this perturbation usually extend beyond the first

boundary lipids but thereafter diminishes (Chapman et al., 1982).

The fluid mosaic model also envisaged an entirely fluid lipid

matrix where all lipids exist above their transition temperatures.

The transition temperature (AT) is that which causes hydrocarbon

chains to pass from a closely packed ordered crystalline (or gel)

state to a disordered liquid-crystalline configuration which is

accompanied by an abrupt rise in heat absorption. It is apparent

that, for each pure phospholipid, the transition occurs at

characteristic temperatures (T .). This temperature increases with

chain length of the fatty-acyl group in the phospholipid

(Michaelson et al., 1974) and with the degree of unsaturation of

the fatty-acyl chain. For a phosphatidylcholine bearing two

saturated C._ chains, T is 1.8°C; one with two saturated C12 l 18chains has a value of 54.9°C. Similarly with a cis double bond

in each chain of the C chain phospholipid, T is lowered to -22°C18 t(Overath and Thilo, 1978). The nature of the phospholipid head

group is also important. Phosphatidylcholine has a bulky

trimethylammonium terminal head group. If the choline head group is

replaced by ethanolamine, which will pack much more closely and in

a less fluid conformation, T^ is raised by 26°C (Stein, 1986).

It is generally accepted that the degree of saturation of

phospholipids affects the fluidity of membranes. Indeed, this has

been supported by experimental data. Membranes rich in saturated

fatty-acyl groups are measurably less fluid than those containing

proportionally fewer saturated fatty-acyl residues (Yau et al.,

1976).

Since each lipid in the bilayer has its own specific transition

temperature and the plasma membrane contains a diversity of

phospholipids, it is most likely that some will be in a fluid state

while others will be in a less mobile rigid formation. Experimental

evidence supports this theory. Phospholipid membrane bilayers are

not universally fluid, but exist in distinct domains of lipid which

are either predominantly in gel or liquid-crystalline form

(Israelachvili, 1978; Karnovsky et al., 1982). Indeed, it is most

likely that phospholipids are distributed asymmetrically between

the inner and outer surfaces of a membrane although it has yet to

be demonstrated in the yeast plasma membrane. Israelachvili (1973)

working with artificial membranes comprising phosphatidylglycerol

and phosphatidylcholine proposes that the asymmetry reduces

electrostatic repulsion between negatively charged phosphatidyl­

glycerol molecules when they are concentrated in the outer layer of

a curved membrane, and that distribution is affected by the

physical shape of the membrane in agreement with data from

Michaelson et al. (1973).

Intrinsic proteins are also likely to influence lipid domains

as they generally partition into the fluid regions (Cullis and de

Kruijff, 1979) and, by influencing lipid-lipid interactions, will

affect the fluidity of the lipid bilayer (Esfahani et al., 1981b).

Rank et al. (1978) demonstrated the regulating effect of intrinsic

proteins on membrane fluidity of plasma membranes isolated from

Sacch. cerevisiae. A low molecular-weight protein was found to be

associated only in high viscosity plasma-membrane vesicles which

were separated from low viscosity vesicles. It was proposed that

the protein probably spans only highly viscous domains in the

membrane.

Another flaw in the fluid mosaic model is the absence of

sterols which are known to contribute to the stability of

membranes. Generally they tend to mobilise lipids in the gel state

and condense those in the liquid-crystalline state (Finkelstein and

Cass, 1967; Demel and de Kruijff, 1976). Sterols have relatively

minute head groups compared to phospholipids, these being hydroxyl

groups attached to a bulky and rigid ringed portion. The hydroxyl

head group orientates itself on the surface of the membrane and the

rigid portion wedges into the hydrophobic region, so that sterols

tend to interact specifically with the fatty-acyl chain region of

phospholipids with minimal interaction with neighbouring

phospholipid headgroups.

The phospholipid head group plays an important role in the

packing arrangement and function (Trivedi et al., 1982) of

membranes and, like any molecule, will be aligned in its stable

conformation. They show a preference towards a highly folded

structure with strong intramolecular hydrogen bonds (Pullman and

Berthod, 1974). It is also believed that the nature of the polar

head group affects the packing of hydrocarbon chains in the body of

the membrane. Dipalmitoylphosphatidylcholine (DPPC) will tilt by

approximately 30° relative to the normal to the plane of a simple

bilayer, whereas hydrocarbon chains of dipalmitoylphosphatidyl-

ethanolamine (DPPE) appear to orientate approximately normal to the

plane of the bilayer (McIntosh, 1980) because of the size and

conformation of the phosphatidylcholine head group (Nagle, 1976).

Plasma Membrane Composition and Diffusion

A considerable amount of literature is concerned with the

distribution and packing arrangement of phospholipids in both

natural and artificial membranes, but there is little available

data on yeasts.

Stratford et al. (1987) suggested that the fluidity of the

plasma-membrane lipids may affect the rate of SO^ uptake arguing

that S'codes ludwigii, being richer in unsaturated phospholipid

fatty-acyl residues, will have a more permeable plasma membrane

than Sacch. cerevisiae. Konttinen and Suomalainen (1977) found that

Sacch. cerevisiae enriched with oleic acid did show increased

permeability to pyruvate compared with cells with more saturated

membranes, and they presumed this was because of increased mobility

of the fatty-acyl groups. Thomas et al. (1978) use a similar

argument in discussing the permeability of yeast plasma-membranes

to ethanol although this paper was later criticized by Jones and

Greenfield (1987). These workers suggest that membrane fluidity

cannot be assumed from the relative saturation of membrane

phospholipids and that these data in isolation are not reliable.

Indeed, this view is supported by Konttinen and Suomalainen (1977)

who saw only a 20% increase in passive diffusion of pyruvate with a five-fold increase in membrane unsaturation in Sacch. cerevisiae.

It is reasonable to assume that carbon chain length and the

degree of saturation of fatty-acyl residues will affect the

geometry of the plasma membrane, but the relative importance of

these factors is unknown. With the current understanding of

membrane structure and function, if the geometry and by inference

the fluidity of the plasma-membrane are altered, then presumably

the diffusion of molecules across that membrane will also be

influenced. Jones and Greenfield (1987) propose that the relative

proportions of the different phospholipids have a considerable

influence upon packing of phospholipids in the membrane because of

the distinctive configuration of the head groups. The alignment of

phospholipid head groups is dependent upon their respective size

and charge (Michaelson et al., 1974; Israelachvili et al., 1980;

Stein, 1986). Sterols are also likely to contribute to the packing

geometry of the plasma membrane. Experimental data have shown that

cholesterol is far more efficient in lowering passive permeability

of phospholipid bilayers than is lanosterol (Yeagle, 1985). Thomas

et al. (1978) showed that the ability of cells to remain viable in

the presence of ethanol shows a marked dependence upon sterol

structure, demonstrating that sterols may regulate membrane

fluidity.

A number of theories have been proposed to account for the

diffusion of small molecules across membranes, and these are

comprehensively reviewed by Lee (1975) and Sha'afi (1981). A most

useful model appears to be that in which the small diffusing

molecule is assumed to dissolve in the bilayer and move across bysksL,

diffusion (Zwolinski^ 1949) where the rate of diffusion is a

function of the solubility of the diffusing molecule in the lipid

bilayer. This is in agreement with "Overton's Rule" (Overton, 1899)

which states that the permeability coefficient of a molecule

passing through a lipid bilayer correlates with its oil/water

partition coefficient. However some very small molecules, e.g.

water, formamide and formic acid, permeate lipid bilayer membranes

faster than predicted by Overton's Rule (Cohen, 1975; Finkelstein,

1976; Walter and Gutknecht, 1984).

Possible explanations for this behaviour include the "mobile

kink" hypothesis where the bilayer is considered to be a slab of

hydrocarbon with transient holes or pockets which open up as the

hydrocarbon chains rotate about saturated C-C bonds (Lieb and

Stein, 1969; Trauble, 1971). Molecules diffuse across the bilayer

by first diffusing into free volumes in the hydrocarbon region

provided by "kinks" in the chains. Then it is proposed that thermal

fluctuation of the hydrocarbon chains serves to carry diffusing

molecules in mobile free volumes across the hydrocarbon phase as

kinks move in waves along the chains. Walter and Gutknecht (1986),

however, have criticised Trauble's mobile kink mechanism since it

does not account for diffusion of larger molecules which tend to

show less size dependence than smaller molecules. Fettiplace and

Haydon (1980) have also pointed out that the degree of disorder in

most bilayers is greater than that assumed in Trauble's model.

Later work (Galey et al., 1973) has shown that there are two

barriers to membrane permeation. One is provided by the water-

membrane interface and one by the membrane interior. However, the

latter is generally regarded as the rate-limiting step. A more

attractive model envisaged by Lee et al. (1974) shows small

molecules first passing through a transient pore into the fluid

part of the hydrocarbon centre and then diffusing through this

region in a pocket of free volume. Another possible explanation for

the high permeabilities of very small molecules is that "transient

aqueous pores" exist in lipid bilayers (Weaver et al., 1984) but

this was also rejected by Walter and Gutknecht (1986) because it

did not account for the high permeabilities of the smallest

molecules.

Walter and Gutknecht (1986) considered the anomalously high

permeability coefficients of very small molecules (M < 50) andrfound that their permeabilities did not correlate with partition

coefficients but were inversely correlated with molecular volumes.

Finkelstein (1976) suggested that size dependency of smaller

molecules could be explained by the Stokes-Einstein model for

diffusion in a liquid where the diffusion coefficient D is

described by:

D = kT/(67rnr)

where r represents the radius of a sphere diffusing in a continuous

fluid, k is the Boltzmann constant, T is the absolute temperature,

n is the coefficient of viscosity and 6Trnr is the factor describing the frictional drag on a sphere moving through a viscous fluid.

However, in the diffusion of molecules across lipid bilayers the

rate of diffusion decreases in value very steeply with molecular

size and does not obey simple Stokesian fluid-dynamics. The

molecular volume dependence of solute permeability suggests that

the membrane barrier behaves more like a polymer network than a

liquid hydrocarbon. Lieb and Stein (1986) propose that the

non-Stokesian movement may be due to the inability of molecules in

the membrane to flow around the diffusing molecules, presumably

because the hydrocarbon chains are anchored at the membrane water

interface. In ideal Stokesian diffusion, membrane lipids would flow

freely around the diffusing molecules. Walter and Gutknecht (1986)

conclude that only the soft polymer model successfully describes

the non-Stokesian diffusion of non-electrolytes. This idea is

consistent with the "solubility-diffusion" model, applicable to

polymers, which describes diffusion within the hydrocarbon chain

region and is represented by the expression:

K D p = mem memmem — 3-----dmem

where P is the permeability coefficient, K and D are the mem mem memaverage partition and diffusion coefficients for the solute in a

membrane interior, and d is the membrane thickness (Diamond andmemKatz, 1974). This model takes into account both the hydrophobicity

dependence and the molecular volume dependence of non-electrolyte

permeability. In keeping with the polymer model, Lieb and Stein

(1986), explain non-Stokesian diffusion in terms of free volume or

holes between which diffusing molecules jump. Since a suitable hole

must have a volume greater than or equal to the diffusing molecule,

and since there will always be more small holes than large holes,

it follows that small molecules will diffuse much more rapidly than

larger ones.

It is assumed that there is a strong correlation between the

permeability of a membrane to non-electrolytes and the membrane

fluidity, and that permeability is a function of the packing of

lipid molecules in the bilayer. Van Zoelen et al. (1978) employed

this correlation to estimate membrane fluidity. The maximum number

of water molecules than can copermeate with thiourea is a function

of packing of the lipids in the bilayer. These workers found that,

in multilamellar liposomes containing 4% phosphatidic acid in

20 mM-glucose, the maximum number of molecules (N ) of water thatmaxcan copermeate with each molecule of solute is dependent on the

packing properties of the lipids and the size of cavities in the

bilayer. When cholesterol is included in the membrane, the value of

r^ax lowered because closer packing of lipids in the presence of

cholesterol results in a decrease in the concentration of cavities

in the bilayer and lower freedom of motion for the fatty-acyl

chains resulting in lower permeability of the bilayers (Bittman and

Blau, 1972). This effect has been observed in many other systems

including membranes of Acholeplasma laidlawii B (McElhaney et al.,

1973).

Some work on natural membranes includes work by Beguinot et al.

(1987) using rat thyroid cells. They found a decreased membrane

fluidity caused by an absolute increase in membrane cholesterol

with an increased cholesterol/phospholipid ratio and an increased

ratio of saturated to unsaturated fatty-acyl residues in membrane

phospholipids. There is a similar correlation with temperature. The

rate of water permeation through lipid bilayers is sharply lowered

below the transition temperature (Blok et al., 1976) because of the

decrease in cavity size, and permeability is increased when the

bilayer is rich in unsaturated phospholipids because of the

increase in cavity size.

McElhaney et al. (1973) were able to show similar results in

membrane lipids of A. laidlawii B cells and synthesised liposomes

(de Gier et al., 1968). These workers also considered the

permeability to non-electrolytes and found a marked dependency on

chemical structure and chain length of fatty-acyl residues

incorporated into lipid membranes. The incorporation of

branched-chain or unsaturated fatty acids, or fatty acids with

short chain lengths, increased membrane fluidity caused either by

interference with hydrocarbon chain packing or by decreasing chain

length both of which lead to increased non-electrolyte

permeability.

Other workers (Singh et al., 1978), who were concerned with the

effect of altered lipid composition on active transport systems in

Candida albicans and Sacch. cerevisiae (Keenan and Rose, 1979),

found that the activity of specific amino-acid carrier systems

could be influenced by the phospholipid and sterol content of

cells. Uratani et al. (1987), working on the leucine transport

system of Pseudomonas aeruginosa, found that the mean fatty-acyl

chain length of membrane phospholipids was important, and suggest

that there exists an optimal bilayer thickness for maximal carrier

activity intimating a close relationship between structure and

function.

The precise nature of diffusion of molecules in lipid bilayers

still needs clarification but it is certain that the specific lipid

structures in a membrane will affect the fluidity of a membrane and

will also affect diffusion of molecules across the membrane.

The two major aims of this project are firstly to investigate

the nature of SO^ resistance in food-spoilage yeasts and to try to

improve our understanding of the mechanisms of this resistance;

secondly, to explain the differential rates of diffusion of SO^

into strains of Sacch. cerevisiae and Zygosacch. bailii with

respect to plasma-membrane composition.

METHODS

ORGANISMS

The yeasts used were Saccharomyces cerevisiae NCYC 431,

Saccharomyces cerevisiae TC8 (Stratford and Rose, 1985), Zygosaccharomyces bailii NCYC 1427 and Zygosaccharomyces bailii

NCYC 563. The strains were maintained at 4°C on slopes containing

(1 ): agar (MYGP) 20 g, glucose 10 g, malt extract 3.0 g, yeast

extract (Lab M) 3.0 g and mycological peptone 0.5 g (Wickerham,

1951).

EXPERIMENTAL CULTURES

Organisms were grown aerobically in medium containing (1 ):

glucose 20 g, (NH^^SO^ 3.0 g, KH^PO^ 3.0 g, yeast extract (Lab M)

1.0 g, CaCl^.PH^O 30 mg and MgSO^.TH^O 30 mg (adjusted to pH 4.0

with HC1). This was the medium used by Stratford and Rose (1986)

and is referred to as Medium A. It is, however, poorly buffered

and, in experiments in which the yeasts were grown in the presence

of sulphite, it was replaced by Medium B which differed from Medium

A in that KH^PO^ was omitted to be replaced by 13.4 g K^HPO^ and

12.9 g citric acid (adjusted to pH 4.0 with citric acid). Under the

conditions used, the pH value of cultures grown using Medium B did

not fall below 4.0. One-litre portions of medium were dispensed

into 2 1 round flat bottomed flasks which were plugged with cotton4wool and sterilized by autoclaving at 6.89 x 10 Pa for 10 min.

Starter cultures (100 ml medium in 250 ml conical flasks) were

inoculated with a pinhead of yeast from a slant culture and

incubated at 30°C for 24 h on an orbital shaker (200 r.p.m.).

One-litre portions of medium were inoculated with portions of

starter culture containing 0.05 mg dry wt Sacch. cerevisiae NCYC

431, 0.5 mg dry wt Sacch. cerevisiae TCS or 1.0 mg dry wt of either

of the Zygosacch. bailii strains and incubated in a constant

temperature (30°C) room with stirring (100 r.p.m.) on a flat-bed

stirrer.

Organisms were grown anaerobically by a modification of the

method of Alterthum and Rose (1973) in medium containing (1 ):

glucose 50 g, KH^PO^ 4.5 g, (NH^J^SO^ 3.0 g, yeast extract (Lab M)

1 g, CaCl^.PH^O 25 mg and MgSO^.TH^O 25 mg (adjusted to pH 4.0 with

HC1). One-litre portions of medium were dispensed into two-litre

round flat-bottomed flasks and sterilized as already described.

Anaerobic conditions were maintained throughout growth by flushing

the flasks with high-purity nitrogen from which the last traces of

oxygen had been removed by a column-type Oxy-Trap (Alltech

Associates Incorporated, Deerfield, Illinois, U.S.A.). Prior to

inoculation, the medium was supplemented with ergosterol (5 mg 1 )

and an unsaturated fatty acid (30 mg 1 ) either myristoleic acid9 9(C, . , - A ), palmitoleic acid (C,_ - - A ), oleic acid 14:1 lo:19 9 12(C - A ), linoleic acid (C - A ’ ), linolenic acidlo:l lo:29 12 15 11(C 0 - A ’ * ) or 11-eicosenoic acid (C__ . - A ). Portions oflo:3 2uil

medium were inoculated with 1 mg dry wt organisms from an overnight starter culture grown in medium B and incubated as previously

described. Control cultures lacking unsaturated fatty acid were

incubated with each batch of experimental cultures. When growth

in the control exceeded 0.1 mg dry wt ml experimental

cultures were discarded. Growth was followed by measuring the

optical density of portions of culture at 600 nm, measurements

being related to dry wt of organism by a standard curve constructed

for each strain of yeast. Organisms were harvested from

mid-exponential phase cultures, containing 0.5 mg dry wt Sacch.

cerevisiae ml or 0.25 mg dry wt of Zygosacch. bailii ml by

filtration through a membrane filter (0.45 ym pore size; 50 mm

diam.; Oxoid) and washed twice with 10 ml 30 mM-citrate buffer (pH

3.0), or by centrifugation (6,000 g, 1 min, 4°C) and washed twice

with distilled water for phospholipid analysis. All centrifugation

regimes were carried out in a Sorvall RC5C refrigerated Superspeed

Centrifuge (Du Pont Company, Wilmington, Delaware, U.S.A.) unless

otherwise stated.

ASSESSMENT OF SULPHUR DIOXIDE TOLERANCE

The ability of yeasts to grow in Medium B containing different

concentrations of sulphite was measured using Dynatech microplates

(Dynatech Laboratories Inc., Alexandria, Virginia, U.S.A.).

Organisms were harvested from mid-exponential phase cultures by

centrifugation (12,000 g for 2 min) and resuspended in fresh medium (pH 4.0) to give 0.1 mg dry wt ml suspension. Using a Digital

Multichannel Pipette (Flow Laboratories) dilute cell suspension

(170 yl) was pipetted into each well of a microtitre plate leaving

one well empty to use as a blank. Sodium metabisulphite (30 yl),

diluted in fresh medium, was added to each well giving final

concentrations of sulphite ranging between zero and 3.3 mM across

the plate. The blank well was filled with 200 yl water and the

plate gently shaken for a few seconds on a Titertek shaker (Flow

Laboratories), to mix the suspensions. Replicate plates were

prepared, covered, sealed in an airtight container with some moist

tissue paper to minimize evaporation and incubated at 30°C on an

orbital shaker (200 r.p.m.). Using a Dynatech Microplate Reader

(MR600), set at 600 nm, optical densities were measured at

intervals up to 6 h after adjusting to zero against the blank well. Cells tended to settle to the bottom of the wells so the plates

were gently agitated before optical density values were measured.

MEASUREMENT OF SULPHITE ACCUMULATION

To measure initial velocities of sulphite accumulation,

organisms grown in Medium A were washed twice with 30 mM-citrate

buffer (pH 3.0) containing 100 mM-glucose, suspended in the same-1buffer at 10 mg dry wt ml and the suspension allowed to

equilibrate for 5 min at 30°C. A reaction mixture consisting of

30 mM-citrate buffer (pH 3.0) containing 100 mM-glucose and35 -110-200 yM-[ SJsulphite (0.20 yCi ml , 1 yCi = 37 KBq) was

prepared in a universal bottle and warmed to 30°C in a water bath.

Radiolabelled sulphite was stored at -20°C in 5 mM-EDTA under

nitrogen gas in 0.5 ml aliquots (0.1 mCi ml ) to prevent

oxidation. Portions (300 yl) of the suspension of organisms were

dispensed into microcentrifuge tubes (Eppendorf). Using a 1.5 ml

multi-dispense syringe pipette, 1.25 ml of radiolabelled sulphite

reaction mixture was added to the organisms and the suspension

quickly mixed by refilling and emptying the syringe. After exactly

4 s, 1.5 ml of the suspension was rapidly filtered through a

membrane filter (0.45 nm pore size; 25 mm diam.; Millipore) which

had been washed with 5 ml 10 mM-sulphite in 30 mM-citrate buffer

(pH 3.0). After filtration, three 1 ml portions of buffered

sulphite solution of the same concentration as employed in the

experiment were used quickly to wash the organisms and filter.

Filters with organisms were then placed in scintillation vials

containing 7 ml Optiphase Safe (Fisons). Radioactivity in the vials

was measured in an LKB Rackbeta liquid scintillation spectrometer

(model 1217).

To measure the extent of sulphite accumulation, washed

organisms grown in Medium A were suspended in glucose-containing

citrate buffer as already described. Radiolabelled sulphite was

added to a 20 ml suspension containing 2 mg dry wt organisms ml

giving final concentrations of 0.1 - 5.0 mM-sulphite (0.2 yCi ml )

and the suspension incubated at 30°C. At appropriate time

intervals, three 1 ml portions of suspension were filtered through prewashed filters as already described. The organisms were washed

with three 1 ml portions of 30 mM-citrate buffer containing

sulphite at the concentrations used in the experiment.

Radioactivity was measured as already described. Background

activity was estimated by repeating the procedure without organisms

to check washing efficiency and to ensure that sulphite was not

binding to filters.

MEASUREMENT OF PLASMA-MEMBRANE AREA IN ORGANISMS

Dimensions of organisms were measured by observation in a light

microscope fitted with an eyepiece graticule. In calculating

membrane areas, it was assumed that organisms of Sacch. cerevisiae

were spheres, those of Zygosacch. bailii were cylinders with

rounded ends and that surface areas were equivalent to

plasma-membrane areas.

MEASUREMENT OF INTRACELLULAR WATER VOLUME

Volumes of intracellular water in organisms in suspension were3calculated by measuring the differential distribution of H^O,

which equilibrates with both extracellular and intracellular water, 14and D-[l- Cjmannitol which is excluded by the plasma membrane.

Initial experiments established that mannitol was not accumulated

by any of the yeasts examined. To do this, washed organisms were

suspended at 10 mg dry wt ml in 30 mM-citrate buffer (pH 3.0)14containing 100 mM-glucose and [ Cjmannitol at 0.01, 1.0 or 100 mM.

The suspensions were incubated for 60 min at 30°C and filtered

through filters that had been prewashed with 5 ml 100 mM buffered

mannitol (0.45 ym pore size; 25 mm diam.; Millipore).

Membranes and organisms were then washed with non-radioactive

mannitol at the concentration used in the experiment, placed in

scintillation vials containing 7 ml Optiphase Safe and

radioactivity measured as already described. To measure the volume

of intracellular water, a suspension of washed organisms (10 mg drywt ml grown in Medium A was prepared and allowed to equilibrate

for 5 min in glucose-containing citrate buffer as already14described. To 15 ml of suspension was added [ Cjmannitol and

tritiated water giving final concentrations of 10 mM- [^C]-1 3 -1mannitol (0.02 yCi ml ) and 0.2 y Ci H^O ml . Suspensions were

incubated with continuous stirring at 4°C for 10 min. Six 1 ml

portions of suspension were then centrifuged in microcentrifuge

tubes (Eppendorf) for 3 min at 12,000 g. Duplicate 200 pi portions

of supernatant from each tube were added to scintillation vials

containing 7 ml Optiphase Safe and radioactivity measured as

previously described. Radioactivity in the suspension of organisms

was measured by placing twelve 200 ul portions of suspension in scintillation vials containing 7 ml Optiphase Safe.

To measure the intracellular water volumes of organisms after

short exposure to sulphite at least 150 mg dry wt organisms were

harvested, washed and suspended in glucose-containing citrate

buffer (pH 3.0) as already described. Sulphite was added to a 75 ml

suspension containing 2 mg dry wt organisms ml giving final

concentrations of 1.0 to 5.0 mM-sulphite. After 10 min incubation

at 30°C with continuous stirring, organisms were centrifuged

(12,000 £ for 2 min) and resuspended in 30 mM-citrate buffer (pH

3.0) containing 100 mM-glucose and 1.0 to 5.0 mM-sulphite at 10 mg

dry wt ml To 15 ml of this suspension was added [ ] mannitol

and tritiated water and intracellular water volumes determined as

already described.

MEASUREMENT OF INTRACELLULAR pH VALUES

(a) Use of Propionic AcidIntracellular pH values of organisms grown in Medium A were

calculated by determining the equilibrium distribution of propionic

acid across the plasma membrane (Conway and Downey, 1950). Washed

organisms, suspended (5 mg dry wt ml *) in 30 mM-citrate buffer

(9 ml) containing 100 mM-glucose (pH 3.0), were allowed to

14equilibrate after adding 1 ml 0.1 mM-[2- Cjpropionic acid

(0.25 yCi ml at 30°C. After 1, 2, 3, 4, 6 and 10 min, duplicate 300 yl portions were taken from the suspension, rapidly filtered

through washed membrane filters (0.45 ym pore size; 25 mm diam.;

Millipore) and washed with 4 x 1 ml 0.01 mM-propionic acid at 4°C.

The filters were transferred, with organisms, to scintillation

vials as already described. Once the time for equilibration had

been ascertained, replicate measurements were obtained by sampling

after 5 min incubation. Intracellular pH values were calculated

from the expression derived by Waddell and Butler (1959):

pH. = pK. + log10 [R(10(pHe_pKe) + 1) - 1]

where R = TA..V /TA .V. , pH. and pH are the internal and external l e e i i epH values, TA. and TA the intracellular and extracellular volumes l eand pK_ and pKg the dissociation constants for propionic acid in

the internal and external environments. The internal and external

dissociation constants for propionic acid were calculated from the

Davies (1962) simplified version of the Debye-Huckel equations.

Values for pK, and pK were calculated to be 4.75 and 4.86, i erespectively.

The effect of the accumulation of sulphite in organisms upon

intracellular pH values was assessed by incubating organisms with

propionic acid as described with the addition of sulphite giving

final concentrations ranging between zero and 5 mM-sulphite,

allowing the sulphite and propionic acid to equilibrate for 10 min, and sampling as already described.

(b) Use of Fluorescein Diacetate as a Fluorescent ProbeThis method relies upon the ability of organisms to take up

non-fluorescing fluorescein diacetate into the cytoplasm and to

enzymically cleave acetate groups through the action of

intracellular esterases to produce fluorescein which is trapped

inside the cell (Slavik, 1982). Fluorescein has a pH-dependent

fluorescence spectrum and so, theoretically, intracellular pH

values can be measured by recording the fluorescence intensities at

520 nm after excitation at 435 nm and 490 nm which are the

positions of the two major peaks in the fluorescence emission

spectrum. A standard curve was constructed by plotting the

fluorescence intensities of fluorescein in 0.1 mM-citrate buffer at

520 nm, after excitation at 435 nm and 490 nm, against pH value

which was varied between pH 2.5 and pH 7.5 by the addition of HC1.

Mid-exponential phase organisms were harvested, washed twice,

resuspended in 30 mM-citrate buffer with 100 mM-glucose (pH 3.0;

10 mg dry wt ml *) and allowed to equilibrate at 30°C. A stock

solution of fluorescein diacetate was prepared (10 mM in acetone)

and kept in the dark to minimise spontaneous decomposition.

Dilutions were prepared only when required. A portion (5 ml) of the

cell suspension was left untreated and used as a blank. The rest of

the suspension was incubated at 30°C for at least 30 min with

100 viM fluorescein diacetate or until there was visible fluorescence. After incubation, the organisms were thoroughly

washed and resuspended in the original volume of buffer, samples

(0.5 ml) were placed in a cuvette of an Amico-Bowman Spectro-

fluorometer (adapted from right angled illumination to 45° to allow

measurement of a dense cell suspension) and the fluorescence

intensity recorded at 520 nm after excitation at 490 nm and 435 nm.

The blanks were analysed similarly and their values subtracted from

the test results. The final emission ratios were used to calculate

intracellular pH values from the standard curve.

VIABILITY MEASUREMENTSViability of yeast populations was measured by staining with

methylene blue (Fink and Kiihles, 1933). Portions of suspensions

(0.5 ml) were removed, filtered through membrane filters (0.45 \im

pore size; 25 mm diam.; Millipore), washed with 3 x 1 ml distilled

water, resuspended in water and after appropriate dilution, mixed

with equal volumes of methylene blue solution (0.01%, w/v, methylene blue in 2%, w/v, sodium citrate). After 5 min incubation

at room temperature, wet preparations were prepared on

haemocytometer slides, and the numbers of live and dead cells

established microscopically in a population of at least 500

organisms. Viable organisms were colourless.

ANALYTICAL METHODS

(a) Free SulphiteThe method of Burroughs and Sparks (1964b) was used to measure

total free sulphur dioxide where:

Free S02 = SO,, + H,,S03 + HSOg" + S0 2"

and with the assumption that dissociation of bound sulphur dioxide

was minimised by decreasing the pH value to 1.5. Portions (5 ml) of

culture filtrate were acidified with 5 ml orthophosphoric acid (25%

v/v) followed by removal of free sulphur dioxide under reduced

pressure (70-80 mm mercury) in a gentle stream of air for 30 min.

Sulphur dioxide was trapped in two absorption tubes each containing

5-10 ml freshly prepared, neutralised 1% (w/v) hydrogen peroxide

solution containing 1% (v/v) Tashiro indicator (2 volumes 0.1%

methyl red plus 1 volume 0.1% methylene blue both in 95% ethanol)

by the reaction:

2H+ + S032- + H202 - H2S04 + H20

The sulphuric acid was titrated to a grey end point with 0.01 M

sodium hydroxide which was standardised with potassium hydrogen

iodate. Blank values were obtained by reconnecting two more

absorption tubes for a further 30 min and titrating as already

described. Titre volumes of blanks were subtracted from the test

values and the concentration of sulphur dioxide calculated by the

relationship:

1 ml 0.01 M-Sodium Hydroxide = 0.32 mg Sulphur Dioxide.

(b) PyruvatePyruvate concentrations present in culture filtrates were

determined using pyruvate test combination kits (Boehringer,

Mannheim, West Germany) according to the method of Czok and

Lamprecht (1974). This method is based on the enzymic conversion of

pyruvate to lactate by lactate dehydrogenase (LDH):

„4ntI „+ LDH _ „AT>+Pyruvate + NADH + H ------ » Lactate + NAD

Oxidation of NADH is proportional to the amount of substrate

converted and is measured spectrophotometrically at 340 nm.

(c) AcetaldehydeThe concentration of acetaldehyde in culture filtrates was

determined using the Boehringer, Mannheim UV-method where both free

and bound acetaldehyde are oxidised in the presence of acetaldehyde

dehydrogenase (Al-DH) by nicotinamide-adenine dinucleotide (NAD+)

to acetic acid:

Acetaldehyde + NAD+ + H20 ■A1~DH > Acetic Acid + NADH + H+

Concentrations of NADH were recorded at 340 nm and the

concentrations of total acetaldehyde calculated and compared with

standards containing 0.5, 2.5 and 4.5 mM-acetaldehyde. Sequential

dilutions of standards were prepared both in the presence and

absence of 5 mM-sulphite. The test kit was found to be sensitive to

concentrations of acetaldehyde between 0.05 and 5 mM and results

were unaffected by the presence of sulphite.

(d) Glycerol

Glycerol concentration in culture filtrates was determined by

an assay kit (Boehringer). The kit contained glycerol kinase, which

catalysed conversion of glycerol into glycerol 3-phosphate and ADP,

pyruvate kinase which catalysed conversion of PEP and ADP to

pyruvate and ATP, and lactate dehydrogenase which calaysed

reduction of pyruvate to lactate generating NAD+. The decline in

concentration of NADH was measured spectrophotometrically at

340 nm, and was stoicheiometrically related to the concentration of

glycerol. Values obtained were corrected for the concentrations of

pyruvate known to be in the culture filtrates.

(e) Ethanol

Ethanol concentrations were determined by gas-liquid

chromatography. A portion (3 ml) of culture filtrate was diluted as

necessary with water. Portions (0.5 ml) of diluted sample were

mixed with equal volumes of 0.2% (v/v) acetone in water, and 1 jjl of solution injected onto the column of a Pye GCD gas chromatograph

fitted with a flame ionization detector (oven temperature 300°C).

The column (1.5 m long, 0.4 cm internal diam.) was packed with

Chromosorb 101 (100/120 mesh) and maintained at 150°C. The

injection temperature was 250°C, and the nitrogen gas carrier flow

rate 40 ml min Standards containing 0.05, 0.10, 0.15 and 0.20%

(v/v) ethanol were run with each batch of samples. The value for

the peak height multiplied by the retention time for samples was

related to ethanol concentration by a standard curve.

LIPID ANALYSIS(a) Lipid Extraction

Pre-washed organisms (250 mg) were mixed with 10 ml 80% ethanol

in a universal bottle and heated at 80°C for 15 min in a water bath

to deactivate lipolytic enzymes and to split lipid protein linkages

(Letters, 1967). The extract was filtered through Whatman no. 44

filter paper and the filtrate stored at -20°C while the residue was

extracted twice with chloroform/methanol (2:1 v/v) for 2 and 1 h, respectively, as it was stirred magnetically on a flat bed stirrer

at room temperature. The three extracts were pooled, washed with

0.25 vol. 0.88% KC1 and the mixture left to separate overnight at

-20°C. The lower organic phase was removed, taken to dryness using

a rotary evaporator, and the residue dissolved in 1 ml light petroleum (b.p. 60-80°C). Extracts, if necessary, were stored under

nitrogen gas at -20°C.

Samples were evaporated under a stream of nitrogen gas until

approximately 100 nl remained and streaked onto a 20 x 20 cm 0.25 mm Silica Gel 60 TLC plate (Merck) using a 50 yl Terumo Micro

Syringe (Terumo Corporation, Tokyo, Japan). On the same plate

standards were streaked containing 1 mg phosphatidylethanolamine, ergosterol and palmitic acid ml * in light petroleum (b.p.

60-80°C). The plate was developed in a light petroleum (b.p.

40-60°C)-diethyl ether-acetic acid (70:30:1, by vol.) solvent

mixture, lipids located by spraying with 0.2% (w/v)2',7'-dichlorofluoroscein in ethanol and the plate viewed under UV

(254 nm) radiation. The phospholipid bands were ringed with a

pencil and the appropriate areas scrapped off the plate and

transferred to 5 ml screw top Reactivials (Pierce Chemical Co.,

Chester, England). At this stage samples were either methylated for

GLC analysis or eluted for quantitation of total phospholipids and

separation into individual phospholipid classes.

(b) Fatty-acyl Composition of Total Cellular PhospholipidsTo determine the fatty-acyl composition of phospholipids,

samples removed from TLC plates were methylated by refluxing with

3 ml borontrifluoride (14% w/v in methanol) for 1 h at 80°C in sealed Reactivials. After cooling, each sample was added to 5 ml of

water in stoppered glass tubes, supplemented with 3 ml petroleum

ether and shaken vigorously. The fatty acid methyl esters were

extracted into the petroleum ether. This extraction procedure was

repeated twice more, the extracts pooled, evaporated to dryness

using a rotary evaporator, dissolved in 1 ml petroleum ether and stored under nitrogen gas at -20°C until they were analysed by GLC.

Fatty acid methyl esters were analysed using a fused capillary

column (25 m length; SGE BP 21) in a Pye Unicam GCD chromatograph

fitted with an SGE on-column adaptor. The injection temperature was

250°C, and the column maintained at 110°C for the first 5 min,

after which the column temperature was raised at the rate of 8°C min until it reached 180°C. The carrier gas was hydrogen flowing

at 6 ml min Percentage fatty-acyl compositions were calculated

using an LDC/Milton Roy integrator.

(c) Fatty-acyl Composition of Individual Phospholipid Classes

For separation of individual phospholipid classes samples were

eluted from the gel with 3 x 3 ml of chloroform-methanol-water

(5:5:1 v/v), followed by 3 ml methanol and finally 3 ml

methanol-acetic acid-water (95:1:5 v/v). The pooled extracts were

evaporated to dryness using a rotary evaporator and taken up into

1 ml chloroform-methanol (2:1 v/v). Samples and standards

containing 1 mg phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine or phosphatidylinositol ml * in light petroleum

(b.p. 60-80°C) were applied to TLC plates as described and

developed in chloroform-methanol-acetic acid-water (120:23:10:4.5

v/v) (Tunbuld-Johansson et al., 1987). Fractions were located as

described and compared with standards for identification. Bands

containing phospholipid classes were scraped off and transferred to

screw top vials. An internal standard of 0.2 mg heptadecanoic acid

(1 mg ml in methanol) was added to each sample before methylation

sind GLC analysis as already described.

(d) Analysis of Total Cellular PhospholipidsTotal cellular phospholipid was determined by assaying the

phosphorus content of the eluted phospholipid band using a

modification of the method of Chen et al. (1956). A small portion

of silica gel was removed from each plate, eluted and used for a

blank while 5 mg, 2.5 mg and 1 mg portions of phosphatidylcholine

were used as controls. Samples containing phosphorus were

evaporated to dryness in standard Kjeldahl digestion tubes and

ashed by adding six drops of concentrated sulphuric acid, and

heating in a Kjeldahl digester (Tecator 1007 Digestion System,

Sweden) at 250°C until white fumes appeared and the samples

blackened. Three drops of 72% perchloric acid were added and

digestion continued for 15 min at 250°C or until digestion was

complete. After cooling water was added and the samples made up to

25 ml in volumetric flasks. Samples and standard solutions of

KH^PO^ containing 1-10 Mg of phosphorus were placed into pyrex

tubes and the volume adjusted to 4 ml with distilled water. To this

4 ml of colour reagent containing 6 N sulphuric acid - 2.5% ammonium molybdate - 10% ascorbic acid - water (1:1:1:2 v/v, prepared fresh each day) was added, and the tubes covered and

incubated at 37°C for 2 h. Absorbance values were measured at

820 nm and compared with reagent blanks, controls and a prepared

standard curve. Values for phosphorus contents were multiplied by

25 to give the total phospholipid content.

MATERIALS

All chemicals used were AnalaR grade or of the highest purity

available commercially. Boron trifluoride, 2',7'-Dichloro-

fluorescein and all lipid standards were purchased from Sigma

Chemical Co. Ltd., Poole, Dorset, England. All radioactively

labelled compounds were obtained from Amersham International,

Amersham, England. Gas-liquid chromatography columns were purchased

from Pye Unicam, Cambridge, England and the packing material was

supplied by Chromatography Services Ltd., Hoylake, Merseyside,

England.

RESULTS

GROWTH OF ORGANISMS UNDER AEROBIC CONDITIONSOrganisms grown aerobically reached mid-exponential phase after

approximately 16 h incubation. The generation time during

exponential growth for Sacch. cerevisiae NCYC 431 was 2 h; Sacch.

cerevisiae TC8, 2 h 10 min; Zygosacch. bailii NCYC 1427, 2 h 30 minand for Zygosacch. bailii NCYC 563, 2 h 20 min. Final growth yield

at stationary phase was approximately 1.7 mg ml for strains of

Sacch. cerevisiae and 2.5 mg ml for Zygosacch. bailii.

Conversion factors used to calculate dry weight of organisms

from optical density measurements (O^goonm^ mid-exponential

phase aerobically-grown organisms were as follows: Sacch.

cerevisiae NCYC 431, 0.58; Sacch. cerevisiae TC8, 0.40; Zygosacch.bailii NCYC 1427, 0.55 and Zygosacch. bailii NCYC 563, 0.58. The

conversion factors are equivalent to values of the gradients

derived from plots of 0D___ against (mg dry wt organisms)ml all600nmof which were linear up to at least 0D„_ 0.6.600nm

Values calculated for cell surface area (Table 3) and

intracellular water volume (Table 4) were found to vary between

different strains of yeast.

EFFECTS OF SULPHITE ON AEROBIC GROWTHSulphite inhibited aerobic growth of all four yeasts at

concentrations up to and including 3.3 mM as assessed by the

microplate method (Fig. 2). Zygosaccharomyces bailii NCYC 563 was

the most sensitive and Sacch. cerevisiae NCYC 431 the least.

68.

Table 3. Cell surface areas of aerobically-grown Saccharomyces

cerevisiae and Zygosaccharomyces bailii estimated from

light-microscope observations. Also indicated are the

number of organisms mg present in mid-exponential phase

cultures from which organisms were taken for cell-surface

area estimation. Values quoted for cell number are the

mean of at least three independent analyses. Surface

areas were calculated from the mean dimensions of at

least sixty organisms.

Organism Number of organisms

mg-1

Surface area of organisms

2 -1 (mm (mg dry wt) )

Saccharomyces cerevisiae NCYC 431 5.25 x 107 2600

Saccharomyces cerevisiae TC8 7.89 x 107 5020

Zygosaccharomyces bailii NCYC 1427 3.56 x 107 3770

Zygosaccharomyces bailii NCYC 563 2.73 x 107 3310

Table 4. Intracellular water volumes of aerobically-grown

Saccharomyces cerevisiae and Zygosaccharomyces bailii

determined as described in the Methods section. Values

quoted are the means of at least three independent

determinations ± SD.

Organism Intracellular Intracellularwater volume water volume

(yl (mg dry wt) 1) (fl)

Saccharomycescerevisiae NCYC 431 1.55 ± 0.15 29.5 ± 2.9

Saccharomycescerevisiae TC8 2.74 - 0.13 34.7 ± 1.6

Zygosaccharomycesbailii NCYC 1427 2.05 ± 0.20 57.6 ± 2.6

Zygosaccharomycesbailii NCYC 563 1.85 ± 0.12 67.6 ± 4.4

Percentage of

growth in

control

wells

70.

Sulphite concn (mM)

Figure 2. Effect of sulphite concentration on growth of

Saccharomyces cerevisiae TC8 (O )» Saccharomyces

cerevisiae NCYC 431 ( • ), Zygosaccharomyces bailii

NCYC 1427 (□) and Zygosaccharomyces bailii

NCYC 563 (I) in Medium B in microtitre wells.

Values quoted are the means of measurements

on eight separate plates. The maximum variation

was ± 10%

ACCUMULATION OF SULPHITE UNDER AEROBIC CONDITIONS

Equilibrium levels for aerobic accumulation of sulphite

equivalents were reached somewhat faster with strains of Sacch.

cerevisiae (Fig. 3) than those of Zygosacch. bailii (Fig. 4)

although all four strains had reached equilibrium levels after 10 min irrespective of the concentration of sulphite. Table 5 lists

intracellular water volumes of aerobically-grown yeasts after short

term exposure to sulphite. Vertical Woolf-Eadie plots (Hofstee,

1959) were obtained with initial velocities of accumulation by all

yeasts suspended in high concentrations of SO^ (Fig. 5). However,

at low concentrations of SO^ especially with Sacch. cerevisiae NCYC

431, there was considerable deviation from the vertical.

EFFECT OF SULPHITE ON YEAST VIABILITY

Organisms grown aerobically in Medium A, harvested and washed

as already described, were allowed to equilibrate in glucose-

containing citrate buffer (pH 3.0). Sulphite was added to

suspensions containing 2 mg dry wt organisms ml giving final

concentrations of 0.1 - 5.0 mM-sulphite and the suspensions

incubated for 10 min at 30°C. All four yeasts maintained 98%

viability after exposure to sulphite concentrations up to and

including 5 mM.

EFFECTS OF SULPHITE UPON INTRACELLULAR pH VALUES

Propionic acid accumulated very rapidly in organisms during the

first few minutes exposure and in strains of both Sacch. cerevisiae

and Zygosacch. bailii equilibrium was reached after 5 min (Fig. 6).

35Figure 3. Time-course for accumulation of [ S] sulphite in (a) Saccharomyces cerevisiae

NCYC 431 and (b) Saccharomyces cerevisiae TC8 suspended in 30 mM-citrate buffer (pH 3.0) at 30°C containing 100 mM-glucose and 0.1 mM (O). 0.5 mM (•),1.0 mM (□), 2.0 mM (■) or 5.0 mM (A) sulphite. Values quoted are the

means of three independent determinations. The maximum variation was ±15%.

a)■PX!a

200

150

(a)

w — in s .oo . E

oco•H-PCOi— IoEPOo<

CO 100-pc0(0>•H3O'0 50

-- -

cP-0 °"

1 _L2 4 6 8

Incubation time (min)10

Figure 3.

-o

2 4 6 8Incubation time (min)

10

f\)

35Figure 4. Time-course for accumulation of [ S] sulphite in (a) Zygosaccharomyces bailii

NCYC 1427 and (b) Zygosaccharomyces bailii NCYC 563 suspended in 30 mM-citrate

buffer (pH 3.0) at 30°C containing 100 mM-glucose and 0.1 mM (O), 0.5 mM (#),

1.0 mM (□), 2.0 mM (■) or 5.0 mM (A) sulphite. Values quoted are the

means of three independent determinations. The maximum variation was ±10%.

200 r-(a)

<D-P•Hx:txr— I3CO

150

100

50

2 4 6 8Incubation time (min)

10

Figure 4.

(b)

I______1_____ I___ I______ I____ I0 2 4 6 8 10

Incubation time (min)

co

Table 5. Intracellular water volume of organisms grown aerobically calculated from the14distribution of radiolabelled [2- C]propionic acid after 10 min equilibration with

sulphite in 30 mM-citrate buffer containing 100 mM-glucose (pH 3.0). Values quoted are

the means of three independent determinations ±SD.

Organism Intracellular water volume (yl (mg dry wt) oforganisms after 10 min equilibration with:-1 mM-sulphite 2 mM-sulphite 5 mM-sulphite

Saccharomyces cerevisiae

Saccharomyces cerevisiae

Zygosaccharomyces bailii

Zygosaccharomyces bailii

NCYC 431 1.45

TC8 2.50

NCYC 1427 1.88

NCYC 563 1.83

± 0.15 1.36 ±

± 0.29 2.89 ±

± 0.12 1.94 ±

± 0.21 1.92 ±

0.29 1.44 ± 0.31

0.15 2.57 ± 0.38

0.41 2.07 ± 0.20

0.31 2.00 ± 0.15

75.

40

iHi

CMI

CO•H-Pcdr—I3e3oocdCMoCA

<hO>1X

30

20

10col

/

f

/

.-65 6

-110 x v (SO^ concn, mM)

Figure 5. Woolfe-Eadie plots for accumulation of molecular

SO^ by Saccharomyces cerevisiae TC8 (O ), Saccharomyces

cerevisiae NCYC 431 ( • ), Zygosaccharomyces bailii

NCYC 1427 (□) and Zygosaccharomyces bailii NCYC 563

(■) suspended in 30 mM-citrate buffer (pH 3.0)

containing 100 mM-glucose at 30°C. Concentrations of

molecular SO^ were calculated from data of King et al.

(1981). Bars indicate SD.

76.

ro•HO(0o•Hco•Ho.oa*o1CMChOCo•H•pajrH I3E3OO<

-P

>>uT300E

0.7

0.6

0.5

0.4

0.3

0.2

0.1

----

I 1 I I L2 4 6 8Incubation time (min)

10

r 14 iFigure 6. Time-course for accumulation of [2- CJpropionic acid by

Saccharomyces cerevisiae NCYC 431 (O )» Saccharomyces

cerevisiae TC8 (•), Zygosaccharomyces bailii NCYC 1427 (□) and Zygosaccharomyces bailii NCYC 563 (■) suspended in

citrate buffer containing 10 yMol [2-^C]propionic acid

at pH 3.0. Values quoted are the means of three

determinations ± SD.

The greater the extent of accumulation of sulphite equivalents, the

larger was the decline in internal pH value (Figs. 7 and 8). Equilibrium accumulation values, and therefore decline in internal

pH values, were smallest for Zygosacch. bailii NCYC 1427 (Fig. 8).Intracellular pH values recorded using the fluorescence probe

technique proved unreliable. The mean intracellular pH value of

Sacch. cerevisiae TC8 in citrate-glucose buffer (pH 3.0) was found to be pH 5.68, this value being the average of three determinations

with a standard deviation of ±0.09. Strains of Zygosacch. bailii

either did not take up the fluorescein diacetate or failed to

cleave the acetate groups even after prolonged incubation (2 h) with the dye. Intensities of fluorescence recorded were

insignificant when compared with blank readings and so it was not

possible to assess intracellular pH values of these organisms.

Fluorescein was rapidly produced in Sacch. cerevisiae NCYC 431 but

equally rapidly leaked from the cells into the surrounding buffer.

Consequently, the emission ratio *490/435 decreased, essentially measuring the pH value of the extracellular buffer.

PRODUCTION OF BINDING COMPOUNDS BY ORGANISMS GROWN AEROBICALLY

IN THE PRESENCE OF SULPHITE

The effect of sulphite on growth of the yeasts in 1 litre

cultures (Medium B) was assessed by adding the compound to

early/mid-exponential phase cultures giving final concentrations of

zero, 1 or 2 mM-sulphite, and measuring the effect on density of

organisms and on concentrations in culture filtrates of

acetaldehyde, ethanol, glycerol, pyruvate and free sulphite over

d)-p•HXQ.fi3CO

, — 1min 2.00 . E'—«H COO -p

cc <Do i—1•H tO•p ><0 •HrH 33 UE 0)3OO<

200 t-

160

120

80

40

0J L

6.8

6.4

6.0

5.6

5.2

4 . 8

<1)3r—i 0) >X<XUflj

VoCflu-pC

4 5 0 1Sulphite concn (mM)

Figure 7. Relationship between extent of accumulation of sulphite equivalents (open symbols) and intracellular

pH (closed symbols) in Saccharomyces cerevisiae TC8 (Oand#), and Saccharomyces cerevisiae NCYC 431 (□andH). Measurements were made after organisms had been suspended in buffer for 10 min. Values

quoted are means of at least three independent determinations. Bars indicate SD. oo

(1)-p•HJZOkt—H310

, nC/3 f'—"Sin S,00 , B

<H COo •pcc 0)o rH•H Cfl-p >0) •H«—1 33 O'£ <03OO<

200

160

120

80

40

J L5 00 21 3 4

-I 6.8

6.4

6.0

5.6

- 5.2

-» 4.8

Sulphite concn (mM)

Figure 8. Relationship between extent of accumulation of sulphite equivalents (open symbols)and intracellular pH (closed symbols) in Zygosaccharomyces bailii NCYC 1427 (A and ▲ ),

and Zygosaccharomyces bailii NCYC 563 (V and ▼ ). Measurements were made after

organisms had been suspended in buffer for 10 min. Values quoted are the means

of at least three independent determinations. Bars indicate SD.

ID

Intracellular

pH value

Figure 9. Effect of supplementing cultures of Saccharomyces

cerevisiae NCYC 431 (a), Saccharomyces cerevisiae TC8 (b), Zygosaccharomyces bailii NCYC 1427 (c) and

Zygosaccharomyces bailii NCYC 563 (d) with sulphite (I,

control; A, 1.0 mM, A, 2 mM) on growth and ethanol formation. Also shown are the effects of these

supplements on concentrations of acetaldehyde (0 )»

glycerol (•) and free sulphite (□) in culture

supernatants. After supplementing cultures with

sulphite, they were observed for a further 6 h. Values quoted are the means of three separate determinations.

The maximum variation in values for concentrations of

acetaldehyde and free sulphite was ±10%; for concentrations of ethanol and glycerol the variation

was ±15%.

80.

-p>>UT3GOE

x:P3SOCJ

2.0

1.0

160

I I L J__I J__II__L

80

0

Cocoor—4ocgjC-pw

0Incubation time (h)

No sulphite 1 mM-sulphite 2 mM-sulphite1 1 2.0coCoo<DT5>>,C0)TDi—I03

P0)o<

GOGOO<DP

JCar HGCO

<ua)G|X|

1.0

— O

I I I I L J I I I I I I I L J I I I I I1

-1 8.0

6 0 3 6 0Incubation time (h)

4.0

Figure 9a.

Glycerol concn

(mM)

81.

tHii—iEP>»U73bOE£POGo

1.0

I— I__1 I_I I I0 3 6

16026C o G O80 O

-J oI I L J I I0 3

Incubation time (h)

o§x:pw

as 9E E—' '—C Co Oc Go Oo oQ) a)T3 p>> •H.C x:<D aT3 rH

•—t 3CO toP<D oO 0)< u

No sulphite 1 mM-sulphite

1.0

I 1___ 1_ _ _ _ I_ _ _ _ i i i i i i J I I1

2 mM-sulphite

L6 0 3 6 0Incubation time (h)

- I 8 . 0

4.0

1 -J I I I 13 6

Figure 9b.

Glycerol concn

(mM)

82.

-p

>>UT3bOex:-p•soGo

2.0

1.0

0

160

I I L J I I I I I L J__I

80

-* 0

sE

O§X-pw

0Incubation time (h)

No sulphite 1 mM-sulphite 2 mM-sulphite

COGOOoT3>>X<DT3i—IOJ-P<DO<

2ECOCoo0)-p•Hx:acH3CO<D<UGUh

-O0 — □ l-r----- "

J I I— I__ I I I I I I I I I I I I6 0 3 6 0 3 60 3

” 18.0

4.0

J 0

Incubation time (h)

Figure 9c.

Glycerol concn

(mM)

Acetaldehyde concn

(mM)

ree sulphite concn

(mM)

83.

Incubation time (h)

2 . 0 r-

1.0

No sulphite 1 mM-sulphite 2 mM-sulphite

i i i i i i - i I I I I I I I

o— o-

n 8.o

4.0

-o-•

J I I I6 0 3 6 0

Incubation time (h)

Figure 9d.

Glycerol concn

(mM)

Figure 10. Effect of supplementing cultures of Saccharomyces

cerevisiae NCYC 431 (a), Saccharomyces cerevisiae TC8 (b)f Zygosaccharomyces bailii NCYC 1427 (c) and

Zygosaccharomyces bailii NCYC 563 (d) with sulphite (O,

control, # , 1.0 mM, □ , 2 mM) on pyruvate

concentrations in culture supernatants. After

supplementing cultures with sulphite, they were observed

for a further 6 h. Values quoted are the means of three separate determinations ± SD.

Pyruvate concn

(mM)

Pyruvate concn

(mM)

84.

(a) (b)

0.4 r

0.3

0.2

0.1

J L4 6 0 2Incubation time (h)

0.3 r-

0.2

(c) (d)

0.1

I I I J L 1 J I

Incubation time (h)

Figure 10.

the following 6 h. Growth of Zygosacch. bailii NCYC 563 was virtually completely inhibited following supplementation of

cultures with 1.0 or 2 mM-sulphite (Fig. 9d). Ethanol production

was also completely inhibited. Even in the supplemented cultures in

which growth was almost completely inhibited, there was a decrease

in the concentration of free sulphite despite a lack of production

of acetaldehyde. Production of glycerol and pyruvate (Fig. lOd),

which was detectable in unsupplemented cultures, was also

completely inhibited. A very similar pattern of response was

observed in cultures of Sacch. cerevisiae TC8 (Fig. 9b). The much greater production of glycerol by this strain in unsupplemented

cultures, which reached a concentration of approximately 7 mM in 6 h cultures, was also completely inhibited by supplementation with

2 mM sulphite. In the presence of 1 mM-sulphite acetaldehyde was

produced resulting in a decline in free sulphite concentration,

there was very limited glycerol produced and a marked decline in

pyruvate production (Fig. 10b). Supplementing cultures of Sacch.

cerevisiae NCYC 431 with 1.0 mM sulphite had no effect on growth or

ethanol production (Fig. 9a) and little effect on pyruvate

production (Fig. 10a). In these cultures, the concentration of free

sulphite declined rapidly, while there was an increase in the

production of glycerol and a rapid appearance of acetaldehyde in

the culture filtrates. When cultures of this yeast were

supplemented with 2.0 mM-sulphite, growth was decreased

considerably and this was accompanied by decreased production of

ethanol, glycerol and pyruvate. However, there was a rapid decline

in the concentration of free sulphite, which was accompanied by a

greater increase in acetaldehyde concentration than was observed in

cultures supplemented with 1.0 mM-sulphite. Cultures of Zygosacch.

bailii NCYC 1427 showed a very similar pattern of responses to

those of Sacch.' cerevisiae NCYC 431 (Figs. 9c, 10c) except that

less glycerol was produced in unsupplemented cultures while

supplementation with 1.0 mM-sulphite lowered glycerol production.

When cultures were observed 24 h after supplementation with

sulphite, only cultures of Zygosacch. bailii NCYC 563 and Sacch.

cerevisiae TC8 containing 2 mM-sulphite failed to grow. All of the other cultures, after prolonged lag phases, eventually underwent

normal exponential growth.

Sulphite concentrations in control flasks containing Medium B

and 1.0 or 2.0 mM-sulphite, after 6 h incubation, decreased by 15.3% and 7.8% respectively (Table 6). Samples analysed immediately after addition of sulphite (T = 0) showed that constituents of

Medium B did not bring about significant binding of free sulphite.

FATTY-ACYL COMPOSITION OF PHOSPHOLIPIDS FROM AEROBICALLY

GROWN YEASTS

The principal fatty-acyl residue in phospholipids from

aerobically-grown strains of Sacch. cerevisiae was C.. ., followed 16:1by C-i- -i anc* (Table 7). In both strains of Zygosacch. bailii,18:1 16 :U ---------- ------C-io o was the major fatty-acyl residue in their phospholipids, lo I dfollowed by C,e , and C,_ _ (Table 7).18:1 16:0

Phospholipid classes were separated on TLC plates into distinct

bands. The Rf values obtained for standard phospholipids were as

follows: phosphatidylethanolamine, 0.64 ± 0.02; phosphatidylserine

0.38 ± 0.05; phosphatidylcholine, 0.27 ± 0.02 and phosphatidyl-

inositol, 0.18 ± 0.02. The values quoted are the mean of six

87.

Table 6. Concentration of free sulphite in control flaskscontaining uninoculated Medium B supplemented with

sulphite and sampled over 6 h while being incubated at 30°C and stirred continually. Values represent the mean

of three determinations. The maximum variation was ±5%.

Concentration of free sulphite (mM) Incubation time in media supplemented with:-

(h) 1 mM-sulphite 2 mM-sulphite

03

6

0.98

0.860.83

2.04

1.861.88

Table 7. Fatty-acyl composition of phospholipids from aerobically-grown strains of

Saccharomyces cerevisiae and Zygosaccharomyces bailii» Values quoted are the

means of three independent determinations ±SD. tr indicates that a trace was

detected, - that none was detected.

Fatty-acyl Fatty-acyl residues (percentage of total) in:-residue

Saccharomyces Saccharomyces Zygosaccharomyces Zygosaccharomycescerevisiae cerevisiae bailii bailiiNCYC 431 TC8 NCYC 1427 NCYC 563

10:0 1.3 + 0.2 tr - -

12:0 1.4 + 0.2 0.7 ± 0.3 - -

14:0 4.1 + 0.4 2.2 ± 0.2 tr -

14:1 1.3 + 0.3 tr tr -

16:0 16.2 + 0.8 17.3 ± 0.3 14.7 ± 0.7 11.1 ± 3.0

16:1 52.2 + 1.7 46.4 ± 1.7 12.2 ± 2.2 9.9 ± 1.9

18:0 1.9 + 0.2 2.7 ± 0.3 6.1 ± 1.1 7.5 ± 1.5

18:1 20.3 + 1.0 30.0 ± 1.5 29.6 ± 2.6 33.0 ± 1.618:2 _ 41.2 3.6 38.4 ± 2.8 oooo

independent experiments ± SD.

Strains of Sacch. cerevisiae were found to contain greater

contents of phospholipid (mg dry wt organisms) * than strains of

Zygosacch. bailii (Table 8). The relative proportions of phosphatidylethanolamine (PE), phosphatidylcholine (PC),

phosphatidylinositol (PI) and phosphatidylserine (PS) differed only

very slightly between the four strains. Phosphatidylcholine was the

most abundant phospholipid followed by PE and PI with less than 10%

as PS. Saccharomyces cerevisiae had a lower proportion of PI and a

higher proportion of PE, compared with strains of Zygosacch. bailii

which had approximately equal contents of these phospholipids. In

addition, Zygosacch bailii NCYC 563 had a slightly higher

proportion of PC than the other three yeasts (Table 8).Values for Amol * for each class of phospholipid in Sacch.

cerevisiae NCYC 431 were very similar to those of Sacch. cerevisiae

TC8 but much lower than those calculated for the Zygosacch. bailii strains. Both Zygosacch. bailii strains had similar Amol * values.

For all yeasts the value for Amol for phosphatidylinositol was

much lower than those calculated for the other phospholipid classes

(Tables 9, 10, 11, 12).

The mean fatty-acyl chain length did not vary between

phospholipid classes in strains of Sacch. cerevisiae (Tables 9 and

10). Phospholipids isolated from strains of Zygosacch. bailii

contained fatty-acyl residues that were longer and more variable in

length compared with Sacch. cerevisiae, where phosphatidylcholine

contained the longest fatty-acyl chains and phosphatidylserine the

shortest (Tables 11 and 12).

Table 8. Total phospholipid content of aerobically-grown strains of Saccharomyces cerevisiae and Zygosaccharomyces bailii and the relative proportions of each phospholipid class, namely

phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and

phosphatidylserine (PS). Values quoted are the means of four independent

determinations ±SD.

Organism Total phospholipid content

(mg(250 mg dryx wt organisms) )

Percentage of the total phospholipid class

PC PE PI PS

Saccharomycescerevisiae NCYC 431 10.39 - 0.92 52.2 ± 2.4 28.9 ± 1.6 11.2 ± 1.9 7.4 ± 1.1

Saccharomyces cerevisiae TC8 9.64 ± 0.42 . 51.0 ± 5.4 31.3 ± 3.5 10.1 ± 3.0 8.4 ± 0.7

Zygosaccharomyces bailii NCYC 1427 7.80 ± 0.64 52.0 ± 1.3 21.0 ± 1.3 20.3 ± 0.7 5.7 ± 0.3

Zygosaccharomyces bailii NCYC 563 8.02 ± 0.33 60.1 ± 1.5 16.2 ± 1.5 17.5 2.7 6.3 ± 0.4

Table 9. Fatty-acyl composition of phospholipid classes in aerobically grown Saccharomyces

cerevisiae NCYC 431. Values quoted are the mean of four independent analyses ±SD. tr

indicates that a trace was detected, - that none was detected. Values for Amol were

calculated as described by Kates and Hagen (1964).

Fatty-acylresidue PC

Percentage of the PE

total phospholipidPI

classPS

12:0 tr tr 6.3 ± 1.4 -

14:0 4.0 ± 0.3 3.1 ± 0.5 8.0 ± 1.7 3.7 ± 0.7

14:1 1.6 ± 0.5 tr tr tr

16:0 21.7 ± 0.8 15.3 ± 1.6 35.1 ± 3.3 23.3 ± 1.7

16:1 53.0 ± 2.2 57.1 ± 1.9 24.5 ± 2.4 41.4 ± 2.4

18:0 3.1 ± 0.3 tr 6.4 ± 1.5 tr

18:1 15.7 ± 1.1 23.5 ± 0.6 15.7 ± 2.7 29.4 ± 1.2

Amol ^ 0.70 ± 0.01 0.81± 0.01 0.40 ±0.04 0.71± 0.02Mean fatty- acyl chain length

16.26± 0.05 16.41± 0.05 16.03 ± 0.38 16.53 ±0.10

Table 10. Fatty-acyl composition of phosholipid classes in aerobically grown Saccharomyces

cerevisiae TC8. Values quoted are the mean of four independent analyses ± SD. tr indicates

that a trace was detected, - that none was detected.

Fatty-acylresidue

PercentagePC

of the total PE

phospholipidPI

classPS

12:0 - tr 3.7 ± 1.0 -

14:0 3.3 ± 1.0 4.2 ± 1.3 4.4 ± 1.0 5.9 ± 2.4

14:1 1.8 ± 0.5 tr tr -

16:0 19.5 ± 1.8 18.1 ± 1.2 34.5 ± 2.3 28.6 ± 1.9

16:1 53.7 ± 2.2 51.9 ± 1.1 23.1 ± 3.6 33.3 ± 2.6

18:0 3.6 ± 0.4 tr 7.2 ± 1.5 tr

18:1 17.9 ± 2.1 25.9 ± 2.9 25.0 ± 3.6 30.0 ± 2.8

Amol 1 0.73 ±0.03 0.78 ±0.03 0.48 ±0.03 0.63 ±0.03

Mean fatty- acyl chain length

16.31 ±0.20 16.46 ±0.34 16.41± 0.11 16.49 ±0.22

Table 11. Fatty-acyl composition of phospholipid classes in aerobically-grown Zygosaccharomyces

bailii NCYC 1427. Values quoted are the mean of three independent analysis ± SD. tr

indicates that a trace was detected.

Fatty-acylresidue PC

Percentage of the total PE

phospholipid class PI PS

16:0 9.7 ± 0.5 2.6 ± 1.3 35.3 ± 1.8 8.6 ± 1.5

16:1 13.1 ± 3.5 36.7 ± 4.9 4.6 ± 1.0 45.0 ± 2.9

18:0 4.1 ± 1.3 tr 11.1 ± 1.5 tr

18:1 23.1 ± 1.8 30.3 ± 1.5 31.5 ± 2.3 32.1 ± 1.7

18:2 49.9 ± 2.5 30.0 ± 4.5 17.5 ± 2.4 13.9 ± 2.2

Amol ^ 1.36 ± 0.03 1.27 ± 0.05 0.71 ± 0.04 1.05 ± 0.06

mean fatty- acyl chain length

17.55 ± 0.25 17.13 ±0.10 17.20 ± 0.05 16.86 ± 0.04

Table 12. Fatty-acyl composition of phospholipid classes in aerobically-grown

Zygosaccharomyces bailii NCYC 563. Values quoted are the mean of three

independent analyses ±SD. tr indicates that a trace was detected.

Fatty-acylresidue PC

Percentage of the total PE

phospholipid class PI PS

16:0 10.3 ± 0.5 3.8 ± 0.8 32.1 ± 2.3 11.1 ± 1.3

16:1 7.4 ± 1.2 32.3 ± 1.2 3.6 ± 0.8 40.9 ± 4.3

18:0 6.8 ± 0.8 tr 14.2 ± 1.9 tr

18:1 32.1 ± 1.7 31.4 ± 2.6 36.0 ± 1.8 35.3 ± 2.618:2 43.2 ± 1.6 32.4 ± 2.3 14.6 ± 2.0 12.4 ± 2.1

Amol ^ 1.26 ± 0.03 1.29 ±0.03 0.69 ±0.04 1.01± 0.07

Mean fatty- acyl chain length

17.61 ±0.04 17.26 ± 0.24 17.21 ±0.06 16.88 ± 0.15

The overall mean fatty-acyl chain length and Amol values

calculated for total phospholipids (Table 13) are higher in strains

of Zygosacch. bailii than those of Sacch. cerevisiae and are

inversely proportional to the permeability coefficient calculated35from the initial rates of diffusion of [ S ] sulphite into

organisms (Fig. 5).

The permeability coefficient is defined as the rate of flow

through a unit area of membrane when the concentration difference

across the membrane is 1.0 M. From Fick's first law of diffusion

the following relationship is derived

v = - C2) (Laidler, 1977)

%

where D is the diffusion coefficient, and C2 are theextracellular and intracellular solute concentrations and I is the

thickness of the membrane. The permeability coefficient is the flux

when - C2 = 1 M so that:-P = D = v

thus the permeability coefficient (P) for S02 diffusing across a membrane is equal to v (S02 concn, M) (Fig. 5).

GROWTH OF SACCHAROMYCES CEREVISIAE NCYC 431 UNDER ANAEROBIC

CONDITIONS

When media were supplemented with ergosterol (5 mg 1 *) and an

unsaturated fatty acid (30 mg 1 ) the generation time of organisms

in the mid-exponential phase of growth was 3 h 30 min reaching a

Table 13. Mean fatty-acyl chain length and degree of unsaturation (Amol of total

phospholipids in yeasts grown aerobically compared with their respective

permeability coefficients for SO^ accumulation calculated from data presented in

the Woolf-Eadie plot (Fig. 5). Values for Amol 1 were calculated as described by Kates and Hagen (1964). Values quoted are the means of at least three

independent analyses ±SD.

Organism Mean fatty-acyl chain length of total phospholipid

_1Value for Amol for total phospholipid

Permeabilitycoefficient

(mm(min)

Saccharomyces cerevisiae NCYC 431

Saccharomyces cerevisiae TC8Zygosaccharomyces bailii NCYC 1427

Zygosaccharomyces bailii NCYC 563

16.02 ± 0.33

16.55 ± 0.04

17.50 ± 0.04

17.44 ± 0.16

0.74 ± 0.02

0.77 ± 0.04

1.24 ± 0.02

1.13 ± 0.02

3.83 ± 0.42

5.42 ± 0.55

1.29 ± 0.21

1.51 ± 0.31

final yield at stationary phase of approximately 1.2 mg mlAnaerobic cultures required a much larger inoculum than those grown

aerobically. Organisms in media supplemented with myristoleic acid

underwent a prolonged lag phase, some 3 h longer than other

anaerobically-grown cultures.

Conversion factors used to calculate dry weight of organisms

from measurements of mid-exponential phase Sacch.600nm ----cerevisiae NCYC 431 grown anaerobically in media supplemented with

ergosterol (5 mg 1 and an unsaturated fatty acid (30 mg 1 *)

were as follows: myristoleic acid 0.63; palmitoleic acid

(C ), 0.65; oleic acid (C ), 0.60; linoleic acid (C._ _),l b :l lo:l 18:2

0.68; linolenic acid (C ), 0.62 and 11-eicosenoic acid (C_ ),18:3 2 0 : l

0.57.

The dimensions of anaerobically-grown Sacch. cerevisiae NCYC

431 were not significantly different from those of organisms of

this strain grown aerobically and were not affected by the nature

of the fatty-acid supplement. Cell-surface areas calculated for

anaerobically-grown Sacch. cerevisiae NCYC 431 using dimensions of

aerobically-grown organisms and the number of organisms mg ^

present in mid-exponential phase cultures are shown in Table 14. As

there is very little variation in the surface areas calculated for

organisms grown under different anaerobic conditions a mean surface 2 -1area of 2150 mm (mg dry wt) is used in subsequent calculations.

FATTY-ACYL COMPOSITION OF PHOSPHOLIPIDS FROM ANAEROBICALLY GROWN YEASTS

Neither strain of Zygosacch. bailii grew anaerobically when

Table 14. Cell-surface areas of anaerobically-grown Saccharomyces

cerevisiae NCYC 431 grown in media supplemented with

ergosterol (5 mg 1 and an unsaturated fatty acid

(30 mg 1 ). Also indicated are the number of organisms

mg present in mid-exponential phase cultures from which

organisms were taken for cell-surface area estimation.

Values quoted for cell number are the mean of at least

three independent analyses while surface areas were

calculated from the mean dimensions of at least sixty

aerobically grown organisms.

Fatty acid supplement Number of organisms-1mg

Surface area of organisms

2 -1 (mm (mg dry wt) )

Myristoleic acid (C„ . „ )14:1Palmitoleic acid

Oleic acid (C1 )lo I 1Linoleic acid (C^^)

Linolenic acid (C )18:311-Eicosenoic acid (^20*1^

4.10 x 10'

4.33 x 10'

4.70 x 10'

4.41 x 10'

4.47 x 10

4.23 x 10

2030

2140

2330

2180

22102090

supplemented with ergosterol and oleic acid either singly or

together. Both Sacch. cerevisiae NCYC 431 and TC8 grew with both

ergosterol and oleic acid, to a lesser extent with just ergosterol

and very little in the presence of only oleic acid. Neither strain

grew significantly in lipid-free anaerobic medium (Fig. 11).

Saccharomyces cerevisiae NCYC 431 was selected to study the manner

in which sulphite transport was affected by the composition of the

fatty-acyl residues in cellular phospholipids. Organisms grown in

the presence of C.. , and C__ , fatty acids led to enrichment in 14:1 16:1residues of these acids to the greatest extent (Table 15).

Enrichment with C , C and C residues was to a lesser la: l 18:2 18:3extent, while that with residues was a mere 13%.

EFFECT OF FATTY-ACYL UNSATURATION AND CHAIN LENGTH ON PERMEATION OF SULPHITE INTO YEASTS

Woolf-Eadie plots of initial rates of sulphite accumulation in

anaerobically-grown Sacch. cerevisiae NCYC 431 gave vertical plots

(Fig. 12). The permeability coefficients differ between organisms

grown in media supplemented with different unsaturated fatty acids.

A plot of permeability coefficient against Amol value for

permeation of sulphite by all four yeast strains showed that the

value for the coefficient was greater the lower the Amol * value

(Fig. 13). Values for permeability coefficient and Amol were

linearly related for Sacch. cerevisiae NCYC 431 enriched in

residues of C, , Cic C. and C__ , and also for this strain 14:1 16:1 18:1 20:1enriched in ^18*2 anc* ^18*3 res^^ues (Fig. 14). However, aplot of permeability coefficient against mean fatty-acyl chain

100.

Incubation time (h)

(c) (d)

th 0D

600

nm o

■--------a________ ■ ■--------- ________ _o 0 L 8- .------ ■ -8 e------= ---- -----0o

---- *---- L J____J W ---- 1---- ____ I J18 20 2216 24 16 18 20 22 24

Incubation time (h)

Figure 11. Time-course of growth of Saccharomyces cerevisiae

TC8 (a), Saccharomyces cerevisiae NCYC 431 (b), Zygosaccharomyces bailii NCYC 1427 (c) and

Zygosaccharomyces bailii NCYC 563 (d) grown

anaerobically at 30°C in Medium C only (O)

or Medium C supplemented with 30 mg oleic acid 1 \

(•), 5 mg ergosterol 1 (□), or with both 5 mg

ergosterol 1 and 30 mg oleic acid 1 (■).

Table 15. Fatty-acyl composition of phospholipids from anaerobically-grown Saccharomyces cerevisiae

NCYC 431 grown in medium supplemented with ergosterol and an unsaturated fatty acid.

Values quoted are the means of three independent determinations ±SD. tr indicates that a

trace was detected, - that none was detected.

Fatty-acyl Percentage composition of fatty-acyl residues in phospholipids from organisms grown anaerobically in media supplemented with:-C14:l C16:l C18:l C18:2 C18:3 C20:l

8:0 , „ 4.010.510:0 tr 3.1±1.3 7.813.8 5.913.4 4.612.7 16.111.612:0 tr 4.4±1.3 7.812.4 4.712.6 4.811.9 17.511.514:0 3.8 ±1.9 7.211.1 15.H2.4 9.812.4 9.812.4 13.411.414:1 52.4 ±2.0 tr tr - - 2.110.616:0 34.0 ±2.0 28.111.7 28.013.6 32.911.1 35.711.4 22.912.616:1 2.1 ±0.3 52.116.1 3.711.6 1.110.6 0.710.4 6.612.518:0 5.6±1.0 4.711.1 tr 4.911.6 5.211.2 2.810.418:1 tr tr 35.616.0 - - 1.510.518:2 - - - 40.916.8 - -18:3 - - - - 38.215.9 -20:1 _ _ _ _ _ 13.H5.6

101

Figure 12. Woolfe-Eadie plots for the accumulation of molecular

SO^ by anaerobically-grown Saccharomyces cerevisiae

NCYC 431 in medium supplemented with ergosterol

(5 mg 1 and 30 mg myristoleic acid (O). palmitoleic

acid (•), oleic acid (□), 11-eicosenoic acid (■), linoleic acid (A) or linolenic acid 1 1 (A).Organisms were suspended in 30 mM-citrate buffer (pH

3.0) containing 100 mM-glucose at 30°C and supplemented

with 50 Mmol, 125 pmol or 250 Mmol sulphite.

Concentrations of molecular SO^ were calculated from

data of King et al. (1981). Bars indicate SD.

**}H-OQC•3CD-ro

v of SO^ accumulation £pmol (mm)~2min

roo coo O U1o“I

CD►33CDOcrH-t-1H*C +*<Oo(D*-bH*oH-CDDc+

ro

co

cn

<ji

or

v of S0o accumulationr _ 2 _ i ipmol (mm) min Jroo coo

To cnoT

CDO

no(D►33CDOO'H**-*H*c+«<iOOCD*-•>H*OH*CDOct

N>

CO

cn

102

Figure 13. Correlation between the permeability coefficient for

SO^ accumulation by organisms and the degree of

unsaturation of fatty-acyl residues in phospholipids

isolated from aerobically-grown Saccharomyces

cerevisiae NCYC 431 (a), Saccharomyces cerevisiae TC8(b), Zygosaccharomyces bailii NCYC 1427 (c),

Zygosaccharomyces bailii NCYC 563 (d) and from

anaerobically-grown Saccharomyces cerevisiae NCYC 431

grown in media supplemented with ergosterol and (i)

myristoleic acid, (ii) palmitoleic acid, (iii) oleic

acid, (iv) linoleic acid, (v) linolenic acid or (vi)

11-eicosenoic acid. Values for Amol were calculated

as described by Kates and Hagen (1964). Values quoted

are the means of three independent determinations ±SD.

103.

Amol

Figure 13.

Relationship between the mean fatty-acyl chain length

and degree of unsaturation (Amol of fatty-acyl

residues in phospholipids isolated from aerobically-

grown Saccharomyces cerevisiae NCYC 431 (a),

Saccharomyces cerevisiae TC8 (b)f Zygosaccharomyces bailii NCYC 1427 (c), Zygosaccharomyces bailii NCYC 563

(d) and from anaerobically-grown Saccharomyces

cerevisiae NCYC 431 supplemented with ergosterol and

(i) myristoleic acid, (ii) palmitoleic acid, (iii)

oleic acid, (iv) linoleic acid, (v) linolenic acid or

(vi) 11-eicosenoic acid. Values for Amol were

calculated as described by Kates and Hagen (1964).

Values quoted are the means of three separate

determinations ±SD.

CO**1H*CQC-}a>

AAro I—

ol\>

Mean fatty-acyl chain length

104

Figure 15. Plot of the permeability coefficient for accumulation

of SO^ and the mean fatty-acyl chain lengths of

phospholipids isolated from aerobically-grown

Saccharomyces cerevisiae NCYC 431 (a), Saccharomyces

cerevisiae TC8 (b), Zygosaccharomyces bailii NCYC 1427 (c), Zygosaccharomyces bailii NCYC 563 (d) and from

anaerobically-grown Saccharomyces cerevisiae NCYC 431

supplemented with ergosterol and (i) myristoleic acid,

(ii) palmitoleic acid, (iii) oleic acid, (iv) linoleic

acid, (v) linolenic acid or (vi) 11-eicosenoic acid. Values quoted are the means of three independent

determinations ±SD.

Mean fatty-acyl chain length

length in phospholipids showed no significant correlation

(Fig. 15).

In all four yeasts there was a very good positive correlation

between values for Amol and mean fatty-acyl chain length of

phospholipids (Table 16, Fig. 14). The correlation coefficient

calculated with eight degrees of freedom was 0.887 which with 99.9%

confidence was very highly significant. There was also a very

significant correlation between the permeability coefficient for

accumulation of SO^ measured in all four strains and the ratio of

the mean fatty-acyl chain lengths and degree of unsaturation

(Amol ) of total phospholipids (Fig. 16). These data had a highly

significant correlation coefficient of 0.791 with 99% confidence

limits.

The total phospholipid content of anaerobically grown Sacch.

cerevisiae NCYC 431 enriched with an unsaturated fatty-acyl residue

was lower than that found in aerobically grown organisms, although

the value was not affected by the nature of the supplement (Table

17). Similarly, proportions of each phospholipid class did not vary

when organisms were grown with different anaerobic supplements,

with one exception. Organisms grown in medium supplemented with

myristoleic acid contained a proportionally larger quantity of

phosphatidylinositol and less phosphatidylcholine compared with

organisms grown with other supplements (Table 17). Only very small

differences were observed when the proportions of phospholipid

classes were compared between aerobically and anaerobically

cultured Sacch. cerevisiae NCYC 431. Aerobically-grown organisms

contained a higher proportion of phosphatidylethanolamine and a

107.

Table 16. Mean fatty-acyl chain length and degree of unsaturation

(Amol of total phospolipids in Saccharomyces

cerevisiae NCYC 431, grown anaerobically in media

supplemented with ergosterol and an unsaturated fatty

acid, compared with permeability coefficients calculated

from data presented in the Woolf-Eadie plots (Fig. 12).

Values for Amol were calculated as described by Kates

and Hagen (1964). Values quoted are the means of at

least three independent analyses ±SD.

Fatty-acylsupplement

Mean : chain total

fatty-acyl length of phospholipid

Value for Amol for total phospholipid

Permeabilitycoefficient

(mm(min) )

C14:l 14.91 + 0.10 0.55 + 0.05 2.23 ± 0.35

C16:1 15.59 + 0.15 0.53 + 0.05 2.85 ± 0.30

C18:! 15.41 + 0.54 0.40 + 0.01 4.48 ± 0.43

C18:2 16.16 + 0.67 0.90 + 0.02 3.07 ± 0.79

C18:3 16.14 + 0.48 1.16 + 0.02 2.42 ± 0.32

C20:l 14.31 + 0.51 0.22 + 0.04 7.04 ± 0.61

Figure 16. Correlation between the permeability coefficient for

accumulation of SO^ and the ratio of mean fatty-acyl

chain lengths and the degree of unsaturation ( mol

of total phospholipids in aerobically-grown

Saccharomyces cerevisiae NCYC 431 (a), Saccharomyces

cerevisiae TC8 (b), Zygosaccharomyces bailii NCYC 1427(c), Zygosaccharomyces bailii NCYC 563 (d) and from

anaerobically-grown Saccharomyces cerevisiae NCYC 431

supplemented with ergosterol and (i) myristoleic acid,

(ii) palmitoleic acid, (iii) oleic acid, (iv) linoleic

acid, (v) linolenic acid or (vi) 11-eicosenoic acid. Values quoted are the mean of three independent

determinations ±SD.

Figure 16.

Permeability coefficient ^mm(min)po go £» (Ji cn '•j co-I - 1------ — 1------- 1--------1 I I

108

Table 17. Total phospholipid contents of anaerobically-grown Saccharomyces cerevisiae NCYC

431 in media supplemented with ergosterol and an unsaturated fatty acid and the

relative proportions of each of the phospholipid classes. Values quoted are the

means of three independent analyses ±SD.

Fatty-acyl Total phospholipid Percentage of the total phospholipid classessupplement content

(mg(250 mg dry ^wt organisms) PC PE PI PS

C14:l 7.72 ± 0.90 46.6 ±1.5 20.8 ± 1.4 29.2 ± 2.2 3.4 + 1.8

C16:l 8.24 ± 0.74 59.2 ± 1.4 21.8 ± 0.4 14.2 ± 2.4 4.9 + 1.7

C18:l 8.58 ± 0.74 56.3 ± 3.7 18.1 ± 1.1 18.2 ± 1.6 7.4 + 2.9

C18:2 8.49 ± 0.46 58.0 ± 1.1 16.9 ± 1.7 19.9 ± 2.1 5.3 + 1.4

C18:3 8.21 ± 0.80 56.5 ± 4.2 16.9 ± 1.3 19.9 ± 3.8 6.7 + 1.8

C20:l 8.80 ± 0.47 51.2 ± 4.2 25.6 ± 4.2 16.5 ± 5.2 6.6 + 0.7

109

lower proportion of phosphatidylinositol compared with those grown

anaerobically (Tables 8 and 17).Values for Amol calculated for phospholipids from

anaerobically-grown Sacch. cerevisiae NCYC 431 differ according to

the nature of the fatty-acid supplement. Within each culture,

Amol values for phosphatidylcholine, phosphatidylethanolamine and

phosphatidylserine are all very similar. Values for Amol * for

phosphatidylinositol are all much lower with the exception of

phospholipids from organisms grown in media supplemented with

myristoleic acid where the Amol values for phosphatidylinositol

are not significantly different (Table 18). Mean fatty-acyl chain

lengths in phospholipid classes gave a similar relationship. Mean

fatty-acyl chain lengths of phospholipids from anaerobically grown

Sacch. cerevisiae NCYC 431 also differ according to the nature of

the fatty-acid supplement. Within each culture, mean fatty-acyl

chain-length values in phospholipid classes are very similar with

the exception of phosphatidylinositol which generally has a lower

mean fatty-acyl chain length. However, in cultures supplemented

with myristoleic acid or palmitoleic acid there was no significant

difference between the mean fatty-acyl chain lengths of_ any of the

phospholipid classes (Table 19).

_1Table 18. Degree of unsaturation (Amol ) of phosphatidylcholine (PC),

phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine

(PS) found in Saccharomyces cerevisiae NCYC 431 grown anaerobically in media

supplemented with ergosterol and an unsaturated fatty acid. Values quoted are

the means of three independent analyses ±SD. Values for Amol values were

calculated as described by Kates and Hagen (1964).

Fatty-acylsupplement PC

Amol value PE PI PS

C14:l 0.53 ± 0.03 0.57 ± 0.03 0.47 ± 0.05 0.45 ± 0.06

C16:l 0.53 ± 0.06 0.62 ± 0.07 0.31 ± 0.07 0.62 ± 0.07

C18:l 0.41 ± 0.04 0.48 ± 0.04 0.17 ± 0.03 0.53 ± 0.07

C18:2 0.83 ± 0.05 0.92 ± 0.10 0.40 ± 0.05 0.81 ± 0.09

C18:3 1.22 ± 0.13 1.22 ± 0.13 0.52 ± 0.10 1.07 ± 0.04

C20:l 0.41 ± 0.08 0.36 ± 0.06 0.12 ± 0.04 0.52 ± 0.03

Ill

Table 19. Mean fatty-acyl chain lengths of each of the phospholipid classes found in

Saccharomyces cerevisiae NCYC 431 grown anaerobically in media supplemented

with ergosterol and an unsaturated fatty acid. Values quoted are the means of

three independent analyses ±SD.

Fatty-acylsupplement PC

Mean fatty- PE

-acyl chain length PI

inPS

C14:l 15.19 + 0.40 14.85 ± 0.10 15.21 ± 0.13 15.20 + 0.31

C16:l 15.74 + 0.20 15.57 ± 0.30 15.77 ± 0.10 15.56 + 0.48

C18:1 15.94 + 0.08 16.11 ± 0.22 14.81 ± 0.35 16.16 + 0.45

C18:2 16.21 + 0.22 16.43 ± 0.34 15.39 ± 0.51 16.36 + 0.26

C18:3 16.50 + 0.09 16.39 ± 0.14 15.46 ± 0.41 16.24 + 0.18

C20:l 15.95 + 0.22 15.23 ± 0.42 14.29 ± 0.63 16.31 + 0.13

112

DISCUSSION

The investigations performed can best be discussed by dividing

them into four broad sections. Firstly, there is screening for

sulphite tolerance in yeasts; secondly, the short-term effect of

exposure of yeasts to sulphite; thirdly, the longer term effects,

up to six hours; and finally the contribution of plasma-membrane

phospholipid composition in the control of diffusion of SO^ into

yeasts.

SCREENING FOR SULPHITE TOLERANCE IN YEASTS

Initially it was necessary to isolate a limited number of

strains that displayed a variety of responses to sulphite; four

were selected. Two strains of Sacch. cerevisiae, selected without

any knowledge of their reaction to sulphite, were used to compare

sulphite resistance with two of Zygosacch. bailii, which have been

reported to be extremely resistant to the compound (Thomas and

Davenport, 1985; Warth, 1985). The first two were Sacch. cerevisiae

NCYC 431, which is a strain originating from a distillery and

having a high tolerance of ethanol (Cartwright £t al., 1986, 1987)

and Sacch. cerevisiae TC8, which is a strain used in cider -making and has been reported to excrete H^S (Stratford and Rose, 1985). It

was surprising, therefore, to find that, of the four strains

examined, one of Sacch. cerevisiae was the most tolerant to

sulphite while a strain of Zygosacch. bailii was the most

sensitive. The availability of authenticated strains of Zygosacch.

bailii is limited. Zygosaccharomyces bailii NCYC 563 was included

in the survey because it has been used in research into sulphite

resistance of spoilage yeasts (Cole et al., 1987). Significantly,

it was the least resistant of the strains examined in the present

study.

INITIAL EFFECTS OF SULPHITE ACCUMULATION IN YEASTS

Sulphur dioxide transportTwo yeasts, namely Sacch. cerevisiae (Stratford and Rose, 1986)

and S'codes ludwigii (Stratford et al., 1987), have been shown to

transport SO^ by free diffusion, based on evidence from vertical

Woolf-Eadie plots. The present report shows that passage of SO^

into strains of Zygosacch. bailii is also by free diffusion. It was

also interesting to note that deviation from the vertical, observed

in the present study with strains of Zygosacch. bailii and

previously with Sacch. cerevisiae TC8 (Stratford and Rose, 1986) and S'codes ludwigii (Stratford £t al., 1987), was very much more

pronounced with Sacch. cerevisiae NCYC 431. This suggests that, at

low concentrations of SO , a facilitated transport system operates,

possibly to transport the HSO^ ion. This proposal is in agreement

with Benitez et al. (1983) and Garcia et al. (1983) who

investigated the possibility of there being such an active

transport system in strains of C. utilis. Selenate-resistant

mutants of C. utilis were shown to have a common transport defect

showing an inability to grow in media with either sulphite,

sulphate or thiosulphate as the sole source of sulphur whereas the

wild type grew with any one of these sources. In addition, the

sulphur oxy-anions sulphite, thiosulphate and dithonate were seen

to inhibit competitively active transport of sulphate in wild-type

strains. Therefore a possible explanation for the biphasic

Woolf-Eadie plots seen in the present study is that the common

active transport system observed in C. utilis may well be the same

as that intimated by Stratford and Rose (1986) and which

predominates at low concentrations of sulphite. As sulphite

concentrations are increased, this system rapidly becomes saturated

and masked by diffusion of higher concentrations of molecular SO .

The importance of diffusion of molecular SO^ into organisms is

often overlooked, especially by experimenters primarily concerned

with active transport systems involving sulphite and related

anions. Tweedie and Segel (1970) recorded the existence of distinct

permeases for sulphite and tetrathionate in Penicillium and

Aspergillus species. However, evidence for a sulphite-specific

permease is still questionable, for the data could equally be

interpreted by simple leakage. All transport studies using

multianionic systems are fraught with problems due to oxidation and

cross reaction of anions. Tweedie and Segel (1970) clearly

recognised these disadvantages but, like Benitez et al. (1983), did

not consider the equilibrium position of sulphite. Wherever HSO^

ions exist in solution some proportion must be present as molecular

SO^ depending on the pH value. Evidence for the accumulation of

sulphite may be misleading in these cases and, in fact, merely

reflect molecular SO^ accumulation. Certainly, in those yeasts that

are a major cause of food-spoilage and from the present data, it

seems likely that diffusion of molecular SO^ is common.

Initial rates of accumulation of SO^ are quoted in this thesis2in units of SO^ accumulated per mm surface area of plasma membrane

per minute which takes into account the different sizes of the

different species of yeast. Estimated cell-surface areas are

assumed to equal plasma-membrane surface areas of organisms.

Individual organisms of Zygosacch. bailii have mean plasma-membrane

surface areas approximately twice that of either Sacch. cerevisiae

strain examined. Therefore, by quoting initial rates of

accumulation in this manner, the data have greater physiological

significance. Similarly, by using intracellular water volume as an

approximation for cytoplasmic volume instead of dry weight,

intracellular concentrations of SO^ are made more meaningful and

may be compared between different yeasts. Intracellular water

volumes of individual organisms of Zygosacch. bailii have a mean

value approximately 90% larger than that of Sacch. cerevisiae.

Intracellular water volumes and intracellular pH valuesIntracellular water volumes were not affected by short-term

exposure to sulphite, which seems to contradict data put forward by

Cole and Keenan (1987) declaring that intracellular water volumes

of yeasts decrease in the presence of acid preservatives. Cole and

Keenan (1987) found that there is an inverse relationship between

protoplast volume and population doubling time, and they proposed

that energy is diverted towards maintenance of intracellular pH

value, so that less energy is available for biosynthesis, resulting

in a slower growth rate and a decrease in protoplast volume.

However, these workers found that there was no simple relationship

between intracellular pH value and doubling time. The present

investigations show that, during short-term exposure to sulphite in

all four yeasts studied, there was no change in protoplast volume

despite retardation of growth. It would appear that these yeasts

are able to maintain their physical condition in the presence of

sulphite in the short term. However, observations were not made on

the condition of subsequent generations when retardation of growth

was evident.

All of the organisms studied were notably resilient toward

sulphite and were able to maintain-viability after short-term

exposure to 2 mM-sulphite. Indeed, even when growth was arrested

and the transmembrane pH gradient severely decreased, organisms

were able to recover and undergo normal exponential growth.

On exposure to sulphite, strains of Sacch. cerevisiae and

Zygosacch. bailii were seen to attain intracellular concentrations

of SO^ exceeding 100 times that outside organisms.Zygosaccharomyces bailii NCYC 563 concentrated SO^ by over 200-fold

in the presence of 0.5 mM-sulphite. If the influx of SO^ is

governed by the intracellular pH value of yeasts and the dynamic2-equilibrium between SO^, HSO^ and S0 , then it should be

possible to predict intracellular concentrations of SO^ (Krebs

et al., 1983). Taking Zygosacch. bailii NCYC 563 as an example,

with an intracellular pH value of approximately 6.4 and an

extracellular pH value of 3.0, 0.002 and 5.6% of free sulphite

exists in the molecular form respectively (King et al., 1981). If

intracellular pH value were the only constraint on influx of SO ,

one would expect to see a 2800-fold concentration of SO^ in these

organisms. Clearly this is never achieved. Cole and Keenan (1986)

found that, in similar experiments with Zygosacch. bailii NCYC 563,

the equilibrium distribution of benzoic acid could not be explained

by the difference in pH value across the plasma membrane. Warth

(1988) observed a similar result when investigating accumulation of

benzoic acid by Zygosacch. bailii. It is reasonable to assume that,

within the cytoplasm, the pH value is not constant and more

probably there exists a complex network of different intracellular

pH values and intracellular weak-acid concentrations within

different sub-cellular organelles and domains.

Other considerations include the presence of both intracellular

and extracellular sulphite-binding compounds. Glucose, in the

extracellular buffer, is known to bind sulphite which acts to lower

the extracellular concentration of sulphite. Indeed, this effect

was seen in control flasks when sulphite (1 mM) was added to medium containing glucose (20 g 1 ) with a pH value of 4.0. It resulted

in a 15.3% decrease in the concentration of free sulphite.

Similarly, Vas (1949) found that, when sulphite (5 mM) was added to

buffer (pH 3.97) containing glucose (50 g 1 ), 29.2% of sulphite

became bound. Over the pH range between 3.0 and 5.5 the value for

the equilibrium constant for the sulphite-glucose complex remains

practically unchanged (Vas, 1949). Therefore a similar pattern of

binding should be observed at a pH value of 3.0. The percentage of

sulphite that becomes bound to glucose will naturally depend on the

concentrations of both glucose and sulphite present but, in the

experimental conditions described, it is unlikely that

sulphite-binding by glucose could account for any more than a 30%

decline in free sulphite concentration. Thus, the predicted

accumulation of sulphite in the example quoted above is at least

2000 times that in the extracellular buffer, which is still unrealistic. It is difficult to explain this paradox. A number of

factors are likely to be involved including the sulphite-binding

capacity of intracellular constituents, production and excretion of

sulphite-binding compounds and the buffering capacity of organisms.

Warth (1988) explains the non-equilibrium uptake of benzoic

acid by postulating an active transport system for the export of

anions. But, if the cytoplasmic pH value is maintained, this

requires continuous and unreasonable energy expenditure. Recently

Cole and Keenan (1987) recorded cytoplasmic pH values of 5.70 and

6.05 for exponential-phase cells of Zygosacch. bailii NCYC 563

where the extracellular media had pH values of 2.8 and 4.5,

respectively. Similarly low intracellular pH values were also found

by using fluorescein fluorescence (Cole and Keenan, 1987). A very

low cytoplasmic pH value would explain the apparently low sulphite

concentrations observed and remove the need for active expulsion of

anions. However, the validity of these pH values is questioned

(Warth, 1988) and is not supported by the present study. The

technique using fluorescein diacetate to measure.intracellular pH

value under the present conditions was found to be wholly

unreliable, and was rejected in favour of the method using

radiolabelled propionic acid.

Each of the four yeasts examined, on exposure to sulphite,

accumulated SO^ rapidly until equilibrium was achieved. The final

intracellular concentrations varied among organisms and are most

likely a function of their intracellular buffering capacities.

Intracellular pH values remain fairly constant in the presence of

low concentrations of sulphite but decline rapidly once these are

raised above 1-mM sulphite. A threshold is reached where organisms

can no longer maintain their intracellular pH value. Buffering

capacity becomes exhausted, and intracellular pH values decline

with the influx and dissociation of more SO^. Notably,

intracellular sulphite concentrations at equilibrium increase

linearly with extracellular sulphite concentration. This is in

keeping with a system of free diffusion until the threshold is

reached when, presumably, buffering capacity is exceeded,

intracellular pH control breaks down resulting in a decline in the

transmembrane pH gradient and dissipation of the proton-motive

force across the plasma-membrane. A result of this would be to

retard or inactivate processes, such as active transport of

solutes, that require energy from the proton-motive force. These

data are consistent with the rapid decrease in the content of ATP

in Sacch. cerevisiae when exposed to sulphite (Schimz and Holzer,

1979; Hinze and Holzer, 1986).

Prakash et al. (1986) found that the decreasing effects on the

intracellular ATP level are synergistically potentiated when

sulphite is added together with either m-chloro-peroxybenzoic acid

(CPBA) or nitrite. The mechanisms involved in the synergistic

action of these glycolytic enzyme inhibitors are not fully

understood, but may prove useful in maximising the antimicrobial

effect of sulphite on yeasts.

There is no direct correlation between concentration of

sulphite after equilibration and tolerance to this preservative,

although Zygosacch. bailii NCYC 1427 is significantly able to

maintain a higher intracellular pH value in the presence of

sulphite than the other yeasts examined, which may be contributory

in its relative resistance. However, this trend does not extend to

Sacch. cerevisiae NCYC 431, the other tolerant strain, or to the

less tolerant strains studied.

In the absence of sulphite, all four yeast strains maintained

intracellular pH values between pH 6.4 and 6.7 when they were

allowed to equilibrate under the conditions described. The two more

tolerant strains, namely, Sacch. cerevisiae NCYC 431 and Zygosacch.

bailii NCYC 1427, maintained intracellular pH values that were

highest in this range. When organisms were exposed to low

concentrations of sulphite (0.1 - 1.0 mM), the less tolerant

strains, Zygosacch. bailii NCYC 563 and Sacch. cerevisiae TC8, showed a greater decline in intracellular pH value than either of

the more tolerant strains which indicates that intracellular pH

control may be important in sulphite resistance.

The ability of yeasts to grow in the presence of sulphite is ~~

primarily a function of their ability to produce acetaldehyde.

However, during the first few minutes of exposure to sulphite, it

appears that the intracellular buffering capacities of different

strains of yeast are important and, in terms of sulphite resistance

in yeasts, this may represent a first line of defence.

The buffering capacity of yeast is largely attributed to their

ability to actively extrude hydrogen ions. The buffering action of

actively excreted metabolites, e.g. carbon dioxide and organic

acids, is thought to contribute only 15 to 40% to the overall

buffering capacity (Sigler et al., 1981b). Active transport of

charged species requires ATPase activity and the presence of

intracellular diffusable anions not only in sufficient quantity but

also of sufficiently high plasma-membrane permeability.

Consequently, their availability could limit the buffering capacity

of the organism. In the future, it would be helpful to find out if

the activity of plasma-membrane ATPase is related to sulphite

tolerance in yeasts and the importance of its role in the recovery

of inhibited yeasts.

LONG-TERM EFFECTS OF SULPHITE Stimulation of acetaldehyde production

The present study revealed a direct correlation between ability

of yeasts to grow in the presence of sulphite and sulphite-induced

production of acetaldehyde which suggests that production of this

sulphite-binding compound contributes significantly to resistance.

It is also noteworthy that the two most sulphite-resistant yeasts

examined, namely Sacch. cerevisiae NCYC 431 and-Zygosacch. bailii

NCYC 1427, are able to produce large amounts of acetaldehyde when

growth and ethanol production were almost completely inhibited by

2.0 mM-sulphite. The data are in agreement with the early findings

of Neuberg and Reinfurth (1919) where, in the presence of sulphite,

acetaldehyde and glycerol were produced in equimolar amounts by

strains of Sacch. cerevisiae. Moreover, the data show for the first

time that this is true also for strains of Zygosacch. bailii.

Production of glycerol by Zygosacch. acidifaciens (now recognised

as Zygosacch. bailii) was reported by Nickerson and Carroll (1945)

but this was demonstrated to arise from the existence of a Neuberg

type III fermentation without addition of sulphite which had

previously only thought to occur under alkaline conditions. The

basic fermentation equation (Neuberg type III) from Freeman and

Donald (1957) is as follows:

2 Glucose -► 2 Glycerol + 1 Acetic acid + 1 Ethanol + 2C0

With all four yeasts studied, there was significant glycerol

production in the absence of sulphite via this fermentation. On

addition of sulphite, the switch to Neuberg's second form of

fermentation is evidently not complete. Generally the theoretical

equimolar production of acetaldehyde and glycerol was not seen.

This failure could be attributed to the fact that normal alcoholic

fermentation and possibly Neuberg's third form of fermentation

continue at decreased rates in the presence of sulphite,

particularly evident with more tolerant strains (Sacch. cerevisiae

NCYC 431 and Zygosacch. bailii NCYC 1427).

Saccharomyces cerevisiae NCYC 431, the most resistant strain

examined, in the presence of 1 mM-sulphite was able to maintain

normal growth and ethanol production while simultaneously producing

additional equimolar amounts of glycerol and acetaldehyde. All of

the other data show that additional acetaldehyde is produced in

favour of ethanol. Pyruvate production was not stimulated by

sulphite in any of the yeasts studied. Its production, like that of

ethanol, is directly correlated with cell growth.

It is also feasible that acetaldehyde might be produced by

yeasts from oxidation of ethanol. Indeed, this has been

commercially exploited to produce acetaldehyde (Wecker and Zall,

1987). Acetaldehyde production was induced by sulphite when meat-

spoilage.yeasts were grown with ethanol and in the absence of

glucose. Acetaldehyde did not accumulate in the absence of sulphite

(Nychas et al., 1988). Under these conditions, ethanol is oxidised

to acetaldehyde and seen to accumulate as an intermediate of

substrate catabolism. Free acetaldehyde is subsequently catabolised

to acetic acid and the acetic acid to acetyl-CoA. NADH is finally

regenerated during oxidative phosphorylation (Pons £t al., 1986).

In the presence of glucose, oxidative phosphorylation is suppressed

and this pathway does not function. Conceivably, NADH could be

regenerated with production of glycerol, but there is no evidence

of this occurring. None of the data presented in this work show a

decrease in ethanol concentration accompanied by glycerol

production.

The ability of yeasts to produce acetaldehyde seems to be the

most important factor enabling them to tolerate sulphite. It is

most likely that the decline in intracellular pH value results when

extracellular and intracellular sulphite-binding capacities are

exceeded, and that tolerance to sulphite is determined by an

organism's ability to withstand both a low intracellular pH value

and to produce acetaldehyde. The reason why yeasts show different

capacities to produce acetaldehyde in the presence of sulphite, and

display different tolerances to this preservative, still remains to

be elucidated.

PLASMA MEMBRANE COMPOSITION AND THE DIFFUSION OF SULPHUR DIOXIDE

INTO YEASTSPlasma-membrane composition of aerobically grown yeasts

Aerobically-grown Sacch, cerevisiae was found to contain

phospholipids that were rich in C1c , and C, residues, with16:1 18:1^16*0 res:*-ues accounting for a minor proportion. Under the same

conditions, strains of Zygosacch. bailii contained phospholipids

with predominantly C and C fatty-acyl residues. These datal o : 1 l o : 2

are in keeping with those of Rattray (1988) who summarises the

fatty-acyl composition of whole-cell lipids as distinct from

phospholipids in these yeasts.

Proportions of the four major classes of phospholipid found in

each yeast strain are broadly similar, but again, there are

striking differences between those of Sacch. cerevisiae and

Zygosacch. bailii. The latter have a higher proportion of

phosphatidylinositol, a lower proportion of phosphatidylcholine and

generally contain less phospholipid compared with strains of Sacch.

cerevisiae. In all four yeasts examined, phosphatidylinositol

contained fatty-acyl residues that were always more saturated than

those found in the other phospholipid classes. This feature is

thought to be of importance because phosphatidylinositol is

recognised as a precursor involved in recently discovered secondary

messenger systems controlling transduction in mammalian cells. In

these cells, phosphatidylinositol is initially phosphorylated to

phosphatidylinositol 4-phosphate and then to phosphatidylinositol

4,5-bisphosphate. Growth factors then, acting via a GTP-binding

protein, stimulate a phosphodiesterase which cleaves phospha-

tidylinositol 4,5-biphosphate to diacylglycerol and inositol

1.4.5-triphosphate. The latter acts to release calcium, while

diacylglycerol stimulates protein kinase C activity and it appears

that both pathways act to control DNA synthesis (Berridge, 1987).

Presently, similar evidence is accumulating for the existence of

such systems in yeasts. The active secondary messenger, inositol

1.4.5-triphosphate, has already been detected in Sacch. cerevisiae

(Kaibuchi ^t al., 1986). Moreover, the loss of radioactivity from

pulse-labelled di- and tri-phosphoinositides in these organisms

demonstrates rapid turnover of these intermediary compoundsa,b

(Steiner and Lester, 1972j) reinforcing their potential role in a

messenger system.

Permeability coefficients derived from the vertical Woolf-Eadie

plots show the two strains of Zygosacch. bailii to have lower

coefficients of SO^ accumulation than either of the Sacch.

cerevisiae strains which focuses ones thoughts on the specific

plasma-membrane composition of each yeast and its contribution in

the regulation of SO^ diffusion. Both strains of Zygosacch. bailii

show a slower rate of accumulation of propionic acid compared to

either strain of Sacch. cerevisiae and, notably, do not accumulate

fluorescein diacetate whereas both Sacch. cerevisiae NCYC 431 and

TC8 readily take up this dye. All of these observations suggest that the plasma membranes of strains of Zygosacch. bailii and

Sacch. cerevisiae have distinctive properties which allow them to

act as selectively permeable barriers to diffusing molecules.

It has been suggested (Stratford et al., 1987) that the degree

of phospholipid unsaturation within a plasma membrane will affect

the degree of fluidity and consequently the permeability

coefficient of SO^ accumulation. However, with the yeast straiins

used in the present study this did not prove to be true. Plasima

membranes of the two strains of Zygosacch. bailii were less

permeable to SO^ despite having a much higher Amol value fo:r

cellular phospholipids compared to either strain of Sacch.

cerevisiae. However, the mean fatty-acyl chain lengths of cellular

phospholipids also varies among organisms and must be taken imto

consideration when describing membrane fluidity. It appears tfriat

Amol * values alone inadequately describe membrane fluidity as they

assume a uniform membrane thickness.

Plasma-membrane composition of anaerobically grown yeastsIn an attempt to separate and assess the contribution of tthe

two variables of fatty-acyl chain length and degree of saturation

of phospholipid fatty-acyl residues to plasma-membrane fluidity,

Sacch. cerevisiae NCYC 431 was grown anaerobically in media

supplemented with ergosterol and specific fatty acids. The airm was

to bring about changes in plasma-membrane composition and therefore

fluidity, and to see if these changes could affect the permeability

to SO^. It is apparent that the two variables are closely link:ed as

one could not be changed without affecting the other. It can b>e

inferred from these findings that there is stringent control oif

plasma membrane synthesis in Sacch. cerevisiae NCYC 431 even w>hen

fatty acids are supplied exogenously.

When Sacch. cerevisiae NCYC 431 was grown anaerobically wiith

different fatty-acid supplements there was no significant chanjge in

the dimensions of the organisms compared with those grown

aerobically. Moreover, although there was a slight decrease in the

number of organisms (mg dry wt) during the mid-exponential phase

of growth when grown anaerobically compared with those grown

aerobically, this was not affected by the nature of the fatty-acid

supplement.

It appears that membrane stability of anaerobically-grown

Sacch. cerevisiae NCYC 431 is maintained by an increased synthesis

of shorter chain fatty-acyl residues, which was observed in

organisms grown in the presence of longer chain unsaturated fatty

acids. The highly significant correlation seen between mean fatty-

acyl chain lengths and values for Amol for cellular phospholipids

indicates that there is very rigid control of membrane fluidity in

organisms. There seems to be a compromise between the requirement

for a fluid membrane and the requirement for a stable bilayer. When

only short-chain unsaturated fatty acids are available, organisms

incorporating these fatty acids also synthesize a higher proportion

of longer chain saturated phospholipids to compensate and to

maintain a normal functional plasma membrane. Similarly, when

organisms sire grown anaerobically in medium supplemented with

long-chain fatty acids ^ 2 0 1 * ^ aPPears "that, with incorporation

of long fatty-acyl residues, shorter residues, possibly originating

from cleavage of long-chain fatty acids, are also incorporated.

The relative extent to which exogenously supplied fatty acids

were incorporated into anaerobically-grown Sacch. cerevisiae NCYC

431 is in general agreement with the results reported by Nes et al.

(1984). The very limited incorporation of residues could be

attributable to the inability of these relatively lengthy residues

to be accommodated into cellular membranes.

Esfahani et al. (1981a) also observed a stringent requirement

for an optimal concentration of saturated fatty-acyl chains with

chain length of C., . and C,_ _ in phospholipids for optimal growth 14:0 16:0of a double-mutant strain of Sacch. cerevisiae. However, no

conclusions were drawn from the relative saturation of cellular

phospholipids in this work.

The strict conservation of membrane fluidity was noted by

Watson and Rose (1980) who proposed that, when Sacch. cerevisiae

NCYC 366 was grown anaerobically, multiply unsaturated fatty acids

are preferentially incorporated into triacylglycerols which are not

membrane components. These workers also suggest that membrane

fluidity could be balanced through synthesis of phosphatidylserine

and phosphatidylinositol which, having a higher proportion of

saturated residues, serve to maintain a degree of rigidity in the

membrane. However, my data do not support this theory as there was

no significant change in the proportions of each phospholipid class

under different anaerobic conditions.

Notably, under anaerobic conditions, exogenously supplied

unsaturated fatty-acyl residues were incorporated preferentially

into phosphatidylcholine, phosphatidylethanolamine and

phosphatidylserine and reflected by the relatively high Amol

values calculated for these phospholipid classes. With the

exception of those organisms grown anaerobically in medium

supplemented with myristoleic acid, phosphatidylinositol extracted

from anaerobically-grown Sacch. cerevisiae NCYC 431 always

contained fatty-acyl residues that were more saturated than those

from the other phospholipid classes. The strong conservation of the

highly saturated form of fatty-acyl residues in phosphatidyl­

inositol, which are synthesized even when fatty acids are supplied

exogenously, gives support to the theory that it is involved in

second messenger systems in these yeasts.

Diffusion of sulphur dioxide and plasma-membrane composition

The permeability coefficient of SO^ accumulation by

anaerobically grown Sacch. cerevisiae NCYC 431 was affected by both

the degree of saturation and mean chain length of phospholipid

fatty-acyl residues but from the initial data it is not possible to

ascertain how each variable has its effect. There is no direct

correlation between mean fatty-acyl chain lengths in cellular

phospholipids and permeability coefficient of SO^ accumulation.

Nevertheless there are two linear relationships seen between values

for Amol calculated for cellular phospholipid fatty-acyl residues

and permeability coefficient of SO^ accumulation. However, a direct

correlation between permeability coefficient of SO^ accumulation

and the ratio of mean fatty-acyl chain lengths and values for

Amol indicates that the most important factor in controlling the

rate of diffusion of SO^ into organisms is membrane thickness, that

is the distance over which diffusing molecules have to travel to

enter the organism. If the mean fatty-acyl chain length is

increased then, assuming a typical fluid mosaic model, the

thickness of the plasma-membrane will also increase and fluidity

will decrease. Membrane thickness will also be dependent on the

presence of perturbing molecules affecting the configuration of the

hydrocarbon regions and on the transition temperature.

Data derived from experiments with both aerobically and

anaerobically-grown yeasts show a good correlation between

permeability coefficient of SO^ accumulation and the ratio of mean

phospholipid fatty-acyl chain length and value for Amol

Generally it is useful to consider aerobically and anaerobically-

grown organisms separately because under anaerobic conditions lipid

composition was artificially altered. However, for analytical

purposes, there is no reason to separate the data. Data derived

from experiments with Sacch. cerevisiae TC8 are consistently different to those derived from those with Sacch. cerevisiae NCYC

431 where one might expect to see better agreement, although they

are well within confidence limits. These discrepancies may arise

from differences in plasma-membrane composition not measured in

this study or from errors most likely derived from estimation in

plasma-membrane surface area. Differences in the physiological

structures of the two strains of Sacch. cerevisiae are supported by

data relating to the number of organisms mg during the mid­

exponential phase of growth which indicate that individual

organisms of Sacch. cerevisiae TC8 are less dense than those of Sacch. cerevisiae NCYC 431.

The chemical features controlling membrane lipid fluidity are,

primarily, the cholesterol/phospholipid ratio, degree of

unsaturation of the phospholipid fatty-acyl chains and the

concentration of membrane proteins (Shinitzky and Yuli, 1982).

However, the value for Amol is generally regarded as an

acceptable, albeit a simplistic, measure of membrane fluidity. It

assumes that the inclusion of double bonds in the hydrocarbon

region of a membrane lipid results in larger gaps in the membrane

because the fatty-acyl chains pack less tightly and allow greater

freedom of motion. Given that diffusing molecules pass through the

plasma membrane via free volumes within the bilayer, as described

in the polymer matrix model in the Introduction, then the higher

density of gaps,in the membrane should, theoretically, allow

diffusion to occur more quickly.

If the molecular packing of the fatty-acyl residues of membrane

phospholipids is considered, a different conclusion may be drawn.

Figure 17 shows a schematic representation of fatty-acyl chains and

how they may be aligned in a membrane. Saturated chains should pack

tightly in a homogeneous bilayer depending upon physical

conditions, e.g. temperature, pressure and pH value. With the

inclusion of one double bond in the chain, the permanent kink not

only inhibits tight packing but also results in shortening the

width of the membrane. The addition of a second double bond causes

the chain to kink again but, because the chain effectively coils

around, it should be able to pack more tightly than the singularly

unsaturated chain. Notably, the second double bond causes a further

decrease in membrane thickness. A third double bond has a similar

effect. Fluidity is not necessarily increased by inclusion of

multiply unsaturated fatty-acyl residues; indeed it may be possible

for these residues to pack more tightly than mono-unsaturated

chains. However, membrane thickness is decreased.

(a) (b) (c) (d)

\H,CC H,CC H,C< H,CC H jC C

HjCC

H jC C

HjC;H jC '

C o *

:C H ,

:C H ,

?c h ,

; c h ,

; c h ,

- C H ,

^CHj> C H ,

\ - o - H,C<' c„,HJC — -CH,h,c^-^ch,H,c/_

H ,C ^ — - C H,

H,C^-3cH,H,C^CH,

CH

IICH

kCH,

\

H C -

HC

H ,C <

H,CCHjCCH ,C <

H— C

H

C — o -

;C H ,

: c h ,

;C H ,

* CHII

-C H

/C H ,

H ,C ^ — .C H ,

H ,C “ CH,

C — ° '

C H ,

^CH,H jC < c h ,

H ,P

HjC

HH C - C

‘ CH

IICH

H C CH,

H ,C -

/C H ,

CH

Figure 17. Space filling models and chemical structures of

fatty acid anions with different numbers of double

bonds: (a) stearic acid; (b) oleic acid; (c) linoleic

acid and (d) linolenic acid. Adapted from

Robertson (1983).

In a membrane under dynamic conditions, free rotation about

single C-C bonds will result in numerous transient gauche and trans

configurations. For example, oleic acid has one permanent kink but,

because of stearic hindrance imposed by adjacent molecules, it is

unlikely it will maintain this configuration and more likely to

rotate to adopt a conformation similar to that given for linoleic

acid (Figure 17). However, the transient existence of the bulky

biphasic molecule does help to explain the effect on permeability

to SO^, and the excellent correlation between permeability

coefficient of SO^ accumulation and the ratio of mean chain length

and Amol value supports the existence of these isomers.

Theories relating to the molecular packing of plasma-membrane

phospholipids raise the question of the validity of the values for

Amol as a measure of membrane fluidity. It seems unlikely that

di- or tri-unsaturated fatty-acyl residues have a two and three

fold effect on increasing membrane fluidity, respectively, compared

with mono-unsaturated residues. In this study, Amol values are

useful to distinguish between the three degrees of unsaturation

because of the different effects on membrane thickness rather than

fluidity. The mean chain-lengths of fatty-acyl residues isolated

from phospholipids in Sacch. cerevisiae NCYC 431 grown

anaerobically in media supplemented with linoleic or linolenic

acids were not significantly different. However, both were longer

than that calculated when this organism was grown under the same

conditions in media supplemented with oleic acid. This suggests

that the former fatty acids have a similar fluidizing or thinning

effect on the plasma membrane which is greater than that imposed by

the incorporation of oleic acid. If fluidity is the primary

criterion controlling incorporation of different fatty-acyl

residues then, implicitly, Amol values are valid parameters of

fluidity for singularly and doubly unsaturated residues but do not

adequately describe fluidity of those membranes containing C18 C 3residues.

The direct correlation between permeability coefficient of SO^

accumulation in yeasts and the ratio of mean fatty-acyl chain

lengths and values for Amol supports the theory that membrane

thickness determines the rate at which a molecule will diffuse

across the yeast plasma membrane. The inclusion of unsaturated

residues results in a shortening of fatty-acyl chains so the ratio

of mean fatty-acyl chain length and value for Amol is

proportional to the plasma-membrane thickness. The result is

clearly seen in Figure 18 where the more fluid region with kinked

fatty-acyl chains results in a narrowing of the membrane. A fully

saturated fatty-acyl chain will be shortened by the equivalent of0

one methyl group in length (1.27 A) and increased in volume from °3about 25 to 50 A when gauche rotamers are formed about two C-C

bonds (Lagaly and Weiss, 1971).

These findings are in parallel with those of de Gier et al.

(1968) and McElhaney et al. (1973) who, working with liposomes,

examined the permeability of glycerol. They found that both by

inclusion of double bonds or by decreasing the chain length of

fatty-acyl residues, permeability was increased. However, in both

cases, it was concluded that the increased permeability can be

simply explained in terms of increased membrane fluidity.

Figure 18. A phospholipid bilayer with a crystalline region (a) where the molecules

lengthen and narrow compared with the adjacent fluid molecules (b)

resulting in a change in membrane thickness. Adapted from Robertson (1983).

I

Figure 18.(b) (a)

136 .

Blok et al. (1975) reported enhanced permeability of liposomes at

the phase-transition temperature to permeating compounds. This is a

generally recognised feature attributed to a sudden increase in

lipid fluidity at the transition temperature. These workers also

noted a strong selectivity with respect to molecular size of the

permeating molecules, and that the extent of permeability depended

strongly on the length of the fatty-acyl chains in saturated

lecithin liposomes (Lenaz, 1979). This finding supports the concept

that, with the formation of more pores in the membrane, solutes

will permeate more quickly and under these conditions fatty-acyl

chain length and hence membrane thickness become the more important

rate-limiting step for solute permeability.

These conclusions must not be considered in isolation. Many

factors are known to influence the fluidity of a membrane and have

not been considered in this Discussion. The packing arrangement of

molecules in the yeast plasma membrane is altered by the proximity

of proteins, sterols and different phospholipids, as well as by

conditions such as temperature and osmotic pressure, all of which

must be considered. This study is confined to the effects of

phospholipids. The nature of the phospholipid head group is known

to affect their arrangement in a bilayer but, as the relative

abundance of each of the four phospholipid classes is very similar

in each of the four strains studied and is not significantly

influenced by inclusion of specific fatty-acyl residues, it is

assumed that their influence is constant as far as these

investigations are concerned. However, the importance of

phospholipid head-group composition in the proper functioning of

the yeast plasma membrane must not be underestimated (Noordam

et al., 1980; Trivedi et al., 1982). Further studies on the

specific supplementation of phospholipids into the yeast plasma

membrane are necessary.

Significantly, lower contents of phospholipid were detected in

anaerobically-grown Sacch. cerevisiae NCYC 431 compared with cells

grown aerobically, which may influence SO^ uptake. Its effect in

isolation is not evident but should be borne in mind. The detailed

analysis of phospholipids in plasma membranes of all four yeast

strains has proved valuable in improving the understanding of

plasma-membrane composition in relation to SO^ permeability but

does not help to explain the toxicity of sulphite. The rate of

diffusion of SO^ into Sacch. cerevisiae NCYC 431 can be changed by

selectively altering the phospholipid composition in the membrane.

However it is unlikely that these changes would be great enough to

affect the overall response to sulphite. If SO^ enters a yeast at a

rate of X mm (min) or at a rate five times this rate, the same

intracellular equilibrium concentration will be ultimately achieved

and the long-term effect will be the same. This is supported by the

non-correlation between permeability to SO^ and resistance in the

four yeast strains studied. It would be interesting to extend this

work to see if specific supplementation in the environment of fatty

acids or sterols affects the inherent ability of a yeast to resist

sulphite. Manipulation of plasma-membrane composition could, by

lowering membrane stability or in some unforeseen way, affect yeast

viability particularly in the presence of sulphite.

Although this work still leaves many avenues of investigation

into the mode of sulphite resistance in yeasts, it is hoped that

the data within will prove instrumental in furthering the present

understanding of the action of sulphite on yeasts. In the context

of the practical application of sulphiting agents in foods and

beverages, the data confirm the importance of excluding possible

sulphite-binding compounds, particularly acetaldehyde from these

products.

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173.

APPENDIX

Included in the appendix is a copy of a paper by B.J.

Pilkington and A.H. Rose published in the Journal of General

Microbiology. This paper contains some of the work presented in

this thesis.

Journal o f General Microbiology ( 1988), 134, 2823- 2830. Printed in Great Britain 2823

Reactions of Saccharomyces cerevisiae and Zygosaccharomyces bailii toSulphite

By B R I D G E T J. P I L K I N G T O N a n d A N T H O N Y H. ROSE*Z y m o l o g y L a b o r a t o r y , S c h o o l o f B i o l o g i c a l S c i e n c e s , B a t h U n i v e r s i t y , B a t h , A v o n B A 2 7 A Y, U K

{ R e c e i v e d 2 2 A p r i l 1 9 8 8 )

Sulphite inhibited growth of all four yeasts studied, Z y g o s a c c h a r o m y c e s bailii N C Y C 563 being most sensitive and S a c c h a r o m y c e s c e r e v i s i a e N C Y C 431 the least. Vertical Woolf-Eadie plots were obtained for initial velocities of 35S accumulation by all four yeasts suspended in high concentrations of sulphite. Equilibrium levels of 35S accumulation were reached somewhat faster with strains of S . c e r e v i s i a e than with those of Z. bailii. With all four yeasts, the greater the extent of 35S accumulation, the larger was the decline in internal pH value. Growth of S . c e r e v i s i a e TC8 and Z. bailii N C Y C 563, but to a lesser extent of S . c e r e v i s i a e N C Y C 431 and Z. bailii N C Y C 1427, was inhibited when mid exponential-phase cultures were supplemented with 1-0 or 2 0 mM-sulphite, the decrease in growth being accompanied by a decline in ethanol production. Unless growth was completely inhibited, the sulphite-induced decline in growth was accompanied by production of acetaldehyde and additional glycerol.

IN T R O D U C T IO N

Sulphite has long been recognized as a powerful antimicrobial agent (Hammond & Carr, 1976). The compound exists in solution in three forms, the proportions of which depend on pH value. At pH values below 1*8, sulphite exists predominantly as free S02 and at pH values above 7*2 largely as SO|_; at intermediate pH values, it exists in various proportions as the bisulphite ion (HSOj; King e t al., 1981). The antimicrobial action of sulphite is greatest at low pH values (Wedzicha, 1984), which explains why the compound is particularly effective against yeasts which, in general, grow best at pH values in the range 3 0-5 0 (Rose, 1987). The greater antimicrobial action of sulphite against S a c c h a r o m y c e s c e r e v i s i a e and S a c c h a r o m y c o d e s l u d w i g i i

at low pH values has been explained by the discovery that, of the three molecular forms in which sulphite exists in solution, only S02 enters these organisms (Stratford & Rose, 1986; Stratford e t al., 1987). Yeast species differ considerably in their ability to resist the antimicrobial action of sulphite. Warth (1985) found that K l o e c k e r a a p i c u l a t a and H a n s e n u l a a n o m a l a were much more sensitive to sulphite than strains of S . c e r e v i s i a e which is generally recognized as being a sulphite- resistant yeast. A yeast which has been reported to be even more resistant to sulphite is Z y g o s a c c h a r o m y c e s bailii (Thomas & Davenport, 1985; Warth, 1985).Little is known of the physiological basis for the different degrees of sulphite resistance among

yeast species. Among strains of S . c e r e v i s i a e , differences in resistance have been attributed to production of compounds, particularly acetaldehyde, that bind sulphite to form a-hydroxysul- phonates (Burroughs & Sparks, 1964), especially when the strains are grown in the presence of sulphite (Rankine, 1968; Rankine & Pocock, 1969; Weeks, 1969). Moreover, Stratford e t al. (1987) attributed the greater sulphite resistance of a strain of S ’c o d e s l u d w i g i i as compared with one of S . c e r e v i s i a e to its ability to produce greater amounts of acetaldehyde. The resistance of S ’c o d e s l u d w i g i i was also caused in part, it was suggested (Stratford e t al., 1987), by its decreased ability to accumulate sulphite. The present paper compares the physiological basis of sulphite resistance in two strains each of S . c e r e v i s i a e and Z. bailii.

0001-4852 © 1988 SGM

2824 B. J. P I L K I N G T O N AND A. H. ROSE

METHODS

Organisms. The yeasts used were S. cerevisiae NCYC 431, S. cerevisiae TC8 (Stratford & Rose, 1985), Z. bailii NCYC 563 and Z. bailii NCY C 1427. They were maintained a t 4 °C on slopes of malt extract-yeast extract- glucose-mycological peptone (M YGP) agar (W ickerham, 1951).

Experimental cultures. Organisms were grown aerobically in a medium containing (l-1): glucose, 20 g; (N H 4)2S 0 4, 3-0 g; K H 2P 0 4, 3-0 g; yeast extract (Lab M), 1-0 g; M gS04 .7 H 20 , 30 mg; and CaCl2.2H 20 , 30 mg (adjusted to pH 4-0 with HC1). This is the medium used by Stratford & Rose (1986) and is referred to as Medium A. It is, however, poorly buffered, and in experiments in which the yeasts were grown in the presence o f sulphite it was replaced by M edium B which differed from Medium A in that K H 2P 0 4 was omitted and replaced by 13-4 g K 2H P 0 4 and 12-9 g citric acid. U nder the conditions used, the pH value of cultures grown using Medium B did not fall below 4-0. Portions of medium (11) were dispensed into 21 round flat-bottomed flasks which were plugged with cotton wool and sterilized by autoclaving a t 6-89 x 104 Pa for 10 min. Starter cultures (100 ml Medium A or B in 250 ml conical flasks) were inoculated with a pinhead of yeast from a slant culture and incubated at 30 °C for 24 h on an orbital shaker (200 r.p.m.). Portions of medium (11) were inoculated with portions of starter culture containing 0-05 mg dry w t S. cerevisiae NCYC 431, 0-5 mg dry wt S. cerevisiae TC8 or 1-0 mg dry wt o f either of the Z. bailii strains. Grow th was followed by measuring the optical density o f portions of culture, measurements being related to dry wt o f organism by a standard curve constructed for each strain o f yeast. Organisms were harvested from mid exponential-phase cultures, containing 0-5 mg dry wt S. cerevisiae ml-1 or 0-25 mg dry wt Z. bailii ml-1, by filtration through a membrane filter (0-45 pm pore size; 50 mm diam .; Oxoid) and washed twice with 10 ml 30 mM-citrate buffer (pH 3-0).

Assessment o f sulphur dioxide tolerance. The ability of the yeasts to grow in Medium B containing different concentrations of sulphite was measured using Dynatech Microplates. Organisms were harvested from mid exponential-phase cultures by centrifugation (12000# for 2 min) and resuspended in fresh medium (pH 4-0) to give a suspension containing 0-1 mg dry w tm l-1 . Cell suspension (170 pi) was pipetted into each well o f a microtitre plate leaving one well empty to use as a blank. Sodium metabisulphite (30 pi) diluted in fresh medium was added to each well giving final concentrations of sulphite ranging between zero and 3-3 m M across the plate. The blank well was filled with 200 pi water and the plate gently shaken for a few seconds to mix the suspensions. Replicate plates were prepared, covered, sealed in an airtight container with some moist tissue paper to minimize evaporation and incubated a t 30 °C on an orbital shaker (200 r.p.m.). Using a Dynatech Microplate Reader (MR600), set at 600 nm, optical densities were measured at intervals up to 6 h after adjusting to zero against the blank well. Cells tended to settle to the bottom of the wells so the plates were gently agitated before optical densities were measured.

Measurement o f sulphite accumulation. To measure initial velocities of sulphite accumulation, organisms grown in M edium A were washed twice with 30 mM-citrate buffer (pH 3-0) containing 100 mM-glucose, suspended in the same buffer at 10 mg dry wt ml-1 and the suspension allowed to equilibrate for 3 m in at 30 °C. A reaction mixture consisting of 30 mM-citrate buffer (pH 3-0) containing 100 mM-glucose and 10-200 pM-[35S]sulphite (0-20 pCi ml-1 ; 1 pCi = 37 kBq) was prepared in a universal bottle and warmed to 30 °C in a water-bath. Labelled sulphite was stored a t —20 °C in 5 mM-EDTA under nitrogen gas in 0-5 ml portions (0-1 mCi ml-1) to prevent oxidation. Portions (300 pi) of the suspension of organisms were dispensed into microcentrifuge tubes (Eppendorf). Using a 1-5 ml multi-dispense syringe pipette, 1-25 ml o f labelled sulphite reaction mixture was added to the organisms and the suspension quickly mixed by refilling and emptying the syringe. After exactly 4 s, 1-5 ml of the suspension was rapidly filtered through a m em brane filter (0-45 pm pore size; 25 mm diam .; Millipore) which had been washed with 5 ml 10 mM-sulphite in 30 mM-citrate buffer (pH 3-0). After filtration, three 1 ml portions of buffered sulphite solution of the same concentration as used in the experiment were used quickly to wash the organisms and filter. Filters with organisms were then placed in scintillation vials containing 7 ml Optiphase Safe (Fisons). Radioactivity in the vials was measured in an LKB Rackbeta liquid scintillation spectrometer (model 1217).

To measure the extent o f sulphite accumulation, washed organisms grown in Medium A were suspended in glucose-containing citrate buffer as already described. Labelled sulphite was added to a suspension containing 2 mg dry wt ml-1 giving a final concentration of 0-1-5-0 mM-sulphite (0-2 pCi ml-1) and the suspension incubated a t 30 °C. A t appropriate time intervals, three 1 ml portions of suspension were filtered through prewashed filters as already described. The organisms were washed with three 1 ml portions o f 30 mM-citrate buffer containing sulphite at the concentration used in the experiment. Radioactivity was measured as already described. Background activity was estimated by repeating the procedure without organisms to check washing efficiency and to make sure that sulphite was not binding to filters.

Measurement o f plasma-membrane area o f organisms. Dimensions o f organisms were measured by observation in a light microscope fitted with an eyepiece graticule. In calculating mem brane areas, it was assumed that organisms of S. cerevisiae were spheres and those of Z. bailii cylinders with rounded ends.

Measurement o f intracellular water volume. Volumes of intracellular water in organisms in suspension were

Reactions o f yeasts to sulphite 2825calculated by measuring the differential distribution o f 3H 20 , which equilibrates with both extracellular and intracellular water, and D-[l-14C]mannitol which is excluded by the plasma membrane. Prelim inary experiments established that m annitol was not accumulated by any of the yeasts examined. To do this, washed organisms were suspended at 10 mg dry wt ml-1 in 30 mM-citrate buffer (pH 3-0) containing 100 mM-glucose and [14C]mannitol at 0-01,1-0 or 100 mM. The suspensions were incubated for 60 min a t 30 °C and filtered through washed membrane filters (0-45pm pore size; 50m m diam .; Oxoid). The membranes were then washed w ith non-radioactive mannitol at the concentration used in the experiment, placed in scintillation vials containing 7 ml Optiphase Safe and radioactivity was measured as already described. To measure the volume of intracellular water, a suspension of washed organisms (10 mg dry wt ml-1) grown in Medium A was prepared as already described. To 15 ml of suspension was added 10 mM-[14C]mannitol (0-02 pCi ml-1) and 0-2 pCi 3H 20 ml-1 . Suspensions were incubated with continuous stirring a t 4 °C for 10 min. Six 1 ml portions of suspension were then centrifuged in microcentrifuge tubes (Eppendorf) for 3 min at 12000 g. Duplicate 200 pi portions of supernatant from each tube were added to scintillation vials containing 7 ml Optiphase Safe and radioactivity was measured as previously described. Radioactivity in the suspension o f organisms was measured by placing 12 200 pi portions of suspension in scintillation vials containing 7 ml Optiphase Safe.

Measurements o f intracellular p H values. Intracellular pH values of organisms grown in Medium A were calculated by determining the equilibrium distribution of propionic acid across the plasma mem brane (Conway & Downey, 1950). W ashed organisms, suspended (5 mg dry wt ml-1) in 30 mM-citrate buffer (9 ml) containing 100 mM-glucose, were allowed to equilibrate after adding 1 ml 0-1 mM-[2-14C]propionic acid (0-25 pCi ml-1) at 30 °C. After 1, 2, 4, 6, 8 and 10 min, duplicate 300 pi portions were taken from the suspension, rapidly filtered through washed mem brane filters (0-45 pm pore size; 25 mm diam .; Millipore) and washed w ith 4 x 1 ml 0-01 mM-propionic acid a t 4 °C. The filters with organisms were transferred to scintillation vials as already described. Once the time for equilibration had been ascertained, replicate measurements were obtained by sampling after 5 m in incubation. Intracellular pH values were calculated from the expression derived by Waddell & Butler (1959):

pHj = pKx + log10[R(lO<pH' “ p*e) + l ) - l]

where R = TAX- V jT A e• Vh pHj and pHe are the internal and external pH values, TAX and TAe the intracellular and extracellular total amounts of propionic acid, V{ and Ve the intracellular and extracellular volumes and pKx and pKt the dissociation constants for propionic acid in the internal and external environments. The internal and external dissociation constants for propionic acid were calculated from the Davies (1962) simplified version of the D ebye- Hiickel equations. Values for pK, and pKt were calculated to be 4-75 and 4-86, respectively.

Analytical methods. Free S 0 2 was assayed by the m ethod o f Burroughs & Sparks (1964), which assumes that dissociation of bound S 0 2 is minimized by lowering the pH value to 1-5. Acetaldehyde, glycerol and pyruvate were determined by using assay kits (Boehringer). E thanol was determined by GLC as described by Beavan et al. (1982).

Chemicals. All reagents used were A nalaR or of the highest grade available commercially. Amersham supplied radioactively labelled chemicals

RESULTS

E f f e c t s o f s u l p h i t e o n g r o w t h

Sulphite inhibited growth of all four yeasts at concentrations up to and including 3-3 mM as assessed by the microplate method (Fig. 1). Z. bailii N C Y C 563 was the most sensitive and S . c e r e v i s i a e N C Y C 431 the least.

A c c u m u l a t i o n o f s u l p h i t e

Vertical Woolf-Eadie plots (Hofstee, 1959) were obtained with initial velocities of accumulation by all yeasts suspended in high concentrations of S02 (Fig. 2). However, at low concentrations of S02 and especially with S . c e r e v i s i a e N C Y C 431, there was considerable deviation from the vertical. Equilibrium levels for accumulation of sulphite equivalents were reached somewhat faster with the strains of S . c e r e v i s i a e than with those of Z. bailii although all four strains had reached these levels after 10 min irrespective of the concentration of sulphite. As suspensions of organisms accumulated equilibrium levels of sulphite equivalents measured after 10 min incubation, intracellular pH values declined (Fig. 3). The greater the extent of accumulation of sulphite equivalents, the larger was the decline in internal pH value. Equilibrium accumulation values, and therefore decline in internal pH values, were smallest for Z. bailii N C Y C 1427 (Fig. 3).

2826 B. J. P I L K I N G T O N A N D A. H. ROSE

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00 1 2 3 4 5 6 7

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Fig. 1. Effect of sulphite concentration on growth of S. cerevisiae TC8 (O), S. cerevisiae N C Y C 431 (#), Z. bailii N C Y C 1427 (□) and Z. bailii N C Y C 563 (■) in Medium B in microtitre wells. Values quoted are the means of measurements on eight separate plates. The maximum variation was ±10%.Fig. 2. Woolf-Eadie plots for accumulation of molecular S 0 2 by S. cerevisiae TC8 (OX S. cerevisiae N C Y C 431 (#), Z. bailii N C Y C 1427 (□) and Z. bailii N C Y C 563 (■) suspended in 30 mM-citrate buffer (pH 3-0) containing 100 mM-glucose at 30 °C. Concentrations of molecular S02 were calculated from the data of King et al. (1981). Bars indicate sd.

Sulphite concn (mM)Fig. 3. Relationship between extent of accumulation of sulphite equivalents (open symbols) and intracellular pH values (closed symbols) in S. cerevisiae TC8 (a), S. cerevisiae N C Y C 431 (b), Z. bailii N C Y C 563 (c) and Z. bailii N C Y C 1427 (d). Measurements were made after organisms had been suspended in buffer for 10 min. Values quoted are means of at least three determinations. Bars indicate SD.

Reactions o f yeasts to sulphite 2827(a)

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Incubation time (h)Fig. 4. Effect of supplementing cultures of S. cerevisiae N C Y C 431 (a) and Z. bailii N C Y C 563 (b) with sulphite (■, control, A> 10 mM, 2 mM) on growth and ethanol formation. Also shown are the effects of these supplementations on concentrations of acetaldehyde (O), glycerol (#) and free sulphite (□) in culture supernatants. After supplementing cultures with sulphite, they were observed for a further 6 h. Values quoted are the means of three separate determinations. The maximum variation in values for concentrations of acetaldehyde and free sulphite was < 10%; for concentrations of ethanol and glycerol the variation was ±15%.

Production o f binding compounds by organisms grown in the presence o f sulphite

The effect o f sulphite on growth o f each o f the yeasts in 1 litre cultures (M edium B) was assessed by adding the compound to mid exponential-phase cultures, and measuring the effect on density o f organisms and on concentrations in culture filtrates o f acetaldehyde, ethanol, glycerol, pyruvate and free sulphite over the following 6 h. Growth o f Z. bailii N C Y C 563 was

2828 B. J. P I L K I N G T O N AND A. H. ROSE

virtually completely inhibited following supplementation of cultures with 1*0 or 2*0 mM-sulphite (Fig. 4 b ) . Ethanol production was also completely inhibited. Even in the supplemented cultures in which growth was almost completely inhibited, there was a decrease in the concentration of free sulphite despite the lack of production of acetaldehyde. Production of glycerol and of pyruvate (not shown), which was detectable in unsupplemented cultures, was also completely inhibited. A very similar pattern of responses was observed in cultures of S . c e r e v i s i a e TC8 (data not shown). The much greater production of glycerol by this strain in unsupplemented cultures, which reached a concentration of approximately 7 m M in 6 h cultures, was also completely inhibited by supplementation with 1-0 or 2-0 mM-sulphite. Supplementing cultures of S .

c e r e v i s i a e N C Y C 431 with 1-0 mM-sulphite had no effect on growth or ethanol production (Fig. 4 a). In these cultures, the concentration of free sulphite declined rapidly, while there was an increased production of glycerol and rapid appearance of acetaldehyde in culture filtrates. When cultures of this yeast were supplemented with 2-0 mM-sulphite, growth was decreased considerably and this was accompanied by decreased production of ethanol and glycerol (Fig. 4 a ) . However, there was again a rapid decline in the concentration of free sulphite, which was accompanied by a greater increase in acetaldehyde concentration than was observed in cultures supplemented with 1 -0 mM-sulphite. Again, production of pyruvate was unaffected (not shown). Cultures of Z. bailii N C Y C 1427 showed a very similar pattern of responses to those of S . c e r e v i s i a e N C Y C 431 (data not shown), except that less glycerol was produced in unsupplement­ed cultures while supplementation with 1-0 mM-sulphite lowered glycerol production.

D ISCUSSION

The two strains of S . c e r e v i s i a e used to compare sulphite resistance with strains of Z. bailii, which have been reported to be extremely resistant to the compound (Thomas & Davenport, 1985; Warth, 1985), were selected without any knowledge of their reaction to sulphite. S .

c e r e v i s i a e N C Y C 431 is a strain originating from a distillery, and has a high tolerance of ethanol (Cartwright e t al., 1986,1987), while S . c e r e v i s i a e TC8 is a strain used in cider-making and which has been reported to excrete H 2S (Stratford & Rose, 1985). It was surprising, therefore, to find that, of the four strains examined, one of S . c e r e v i s i a e was the most tolerant to sulphite while a strain of Z. bailii was the most sensitive. The availability of authenticated strains of Z. bailii is limited. Z. bailii N C Y C 563 was included in the survey because it has been used in research into sulphite resistance of spoilage yeasts (Cole e t al., 1987). Significantly, it was the least resistant of the strains examined in the present study.Two yeasts, namely S . c e r e v i s i a e (Stratford & Rose, 1986) and S ’c o d e s l u d w i g i i (Stratford e t al.,

1987), have been shown to transport S02 by free diffusion, based on evidence from vertical Woolf-Eadie plots. The present report shows that passage of S02 into strains of Z. bailii is also by free diffusion. It was also interesting to note that the deviation from verticality, observed in the present study with strains of Z. bailii and previously with S . c e r e v i s i a e TC8 (Stratford & Rose, 1986) and S ’c o d e s l u d w i g i i (Stratford e t al., 1987), was very much more pronounced with S . c e r e v i s i a e N C Y C 431. This suggests that, at low concentrations of S02, a facilitated transport system operates, possibly to transport the HSOj ion. With vertical Woolf-Eadie plots, the value at the intercept on the abscissa is equivalent to the permeability coefficient for passage of S02 into the organism (Laidler, 1977). It is clear, therefore, that the two strains of Z. bailii have lower permeability coefficients than either of the S . c e r e v i s i a e strains.Our discovery of a correlation between ability of yeasts to grow in the presence of sulphite and

sulphite-induced production of acetaldehyde suggests that production of this sulphite-binding compound contributes significantly to the resistance. It is also noteworthy that the two most sulphite-resistant yeasts examined, namely S . c e r e v i s i a e N C Y C 431 and Z. bailii N C Y C 1427, are able to produce large amounts of acetaldehyde when growth was almost completely inhibited by 2-0 mM-sulphite. Excretion of acetaldehyde together with glycerol in cultures of S . c e r e v i s i a e supplemented with sulphite has been known for many years (Neuberg & Reinfurth, 1918,1919), and constitutes Neuberg’s second form of fermentation (Nord & Weiss, 1958). Our data are in general agreement with the finding of Neuberg & Reinfurth (1919) that, in the presence of

Reactions o f yeasts to sulphite 2829sulphite, acetaldehyde and glycerol are produced in equimolar amounts by strains of S . cerevisiae. Moreover, the data show for the first time that this is true also for strains of Z. bailii. Production of glycerol by Z. a c i d i f a c i e n s (now recognized as Z. bailii) was reported by Nickerson & Carroll (1945).When S02 enters the yeast cell, it encounters an environment which is around pH 6-5 with the

result that a large proportion of the S02 is converted into HSOj. This explains the ability of yeasts to concentrate sulphite intracellularly. At the same time, the intracellular pH value declines, which in turn lowers the transmembrane pH gradient and hence dissipates the proton- motive force across the plasma membrane. A result of this would be to retard or inactivate processes, such as active transport of solutes, that require energy from the proton-motive force. The discovery that the decrease in internal pH value following accumulation of sulphite is not of the same magnitude in all strains of yeast suggests that the internal buffering capacity of organisms might be important in sulphite resistance. While invoking a role for energy metabolism in sulphite resistance of yeasts, it is worth noting that exposure of S . c e r e v i s i a e to sulphite leads to a rapid decrease in the content of ATP (Schimz & Holzer, 1979) which has been attributed primarily to the action of sulphite on the enzyme glyceraldehyde-3-phosphate dehydrogenase (Hinze & Holzer, 1986).

The research reported in this paper was generously supported by the AFRC. We also thank Jill Calderbank for advice.

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